AU4079799A - Transgenic animals produced by homologous sequence targeting - Google Patents

Description

WO 99/60108 PCTIUS99/10731 TRANSGENIC ANIMALS PRODUCED BY HOMOLOGOUS SEQUENCE TARGETING FIELD OF THE INVENTION The invention relates to methods for targeting an exogenous polynucleotide or exogenous 5 complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a target cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active 10 target cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active living target cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell. The invention also relates to compositions and formulations that contain exogenous targeting polynucleotides, complementary pairs of exogenous targeting polynucleotides, chemical substituents of such polynucleotides, and recombinase proteins, 15 including recombinosome proteins and other targeting proteins, used in the methods of the invention. BACKGROUND Homologous recombination (or general recombination) is defined as the exchange of homologous segments anywhere along a length of two DNA molecules. An essential feature of general recombination is that the enzymes responsible for the recombination event can presumably use any 20 pair of homologous sequences as substrates, although some types of sequence may be favored over others. Both genetic and cytological studies have indicated that such a crossing-over process occurs between pairs of homologous chromosomes during meiosis in higher organisms. Alternatively, in site-specific recombination, exchange occurs at a specific site, as in the integration of phage 8 into the E. coli chromosome and the excision of 8 DNA from it. Site-specific recombination 25 involves specific sequences of the phage DNA and bacterial DNA. Within these sequences there is WO 99/60108 PCT/US99/10731 only a short stretch of homology necessary for the recombination event, but not sufficient for it. The enzymes involved in this event generally cannot recombine other pairs of homologous (or nonhomologous) sequences, but act specifically on the particular phage and bacterial sequences. Although both site-specific recombination and homologous recombination are useful mechanisms for 5 genetic engineering of DNA sequences, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence. Site-specific recombination hag been proposed as one method to integrate transfected DNA at chromosomal locations having specific recognition sites (O'Gorman et al. (1991) Science 251: 1351; Onouchi et al. (1991) Nucleic 10 Acids Res. 19: 6373). Unfortunately, since this approach requires the presence of specific target sequences and recombinases, its utility for targeting recombination events at any particular chromosomal location is severely limited in comparison to targeted general recombination. For these reasons and others, targeted homologous recombination has been proposed for treating human genetic diseases. Human genetic diseases include (1) classical human genetic diseases 15 wherein a disease allele having a mutant genetic lesion is inherited from a parent (e.g., adenosine deaminase deficiency, sickle cell anemia, thalassemias), (2) complex genetic diseases like cancer, where the pathological state generally results from one or more specific inherited or acquired mutations, and (3) acquired genetic disease, such as an integrated provirus (e.g., hepatitis B virus). .However, current methods of targeted homologous recombination are inefficient and produce desired 20 homologous recombinants only rarely, necessitating complex cell selection schemes to identify and isolate correctly targeted recombinants. A primary step in homologous recombination is DNA strand exchange, which involves a pairing of a DNA duplex with at least one DNA strand containing a complementary sequence to form an intermediate recombination structure containing heteroduplex DNA (see, Radding, C.M. (1982) Ann. 25 Rev. Genet. 16: 405; U.S. Patent 4,888,274). The heteroduplex DNA may take several forms, including a three DNA strand containing triplex form wherein a single complementary strand invades the DNA duplex (Hsieh et al. (1990) Genes and Development 4: 1951; Rao et al., (1991) PNAS 88:2984)) and, when two complementary DNA strands pair with a DNA duplex, a classical Holliday recombination joint or chi structure (Holliday, R. (1964) Genet. Res. 5: 282) may form, or a double-D 30 loop ("Diagnostic Applications of Double-D Loop Formation" U.S.S.N. 07/755,462, filed 4 September 1991, which is incorporated herein by reference). Once formed, a heteroduplex structure may be resolved by strand breakage and exchange, so that all or a portion of an invading DNA strand is spliced into a recipient DNA duplex, adding or replacing a segment of the recipient DNA duplex. Alternatively, a heteroduplex structure may result in gene conversion, wherein a sequence of an 2 WO 99/60108 PCT/US99/10731 invading strand is transferred to a recipient DNA duplex by repair of mismatched bases using the invading strand as a template (Genes, 3rd Ed. (1987) Lewin, B., John Wiley, New York, NY; Lopez et al. (1987) Nucleic Acids Res. 15: 5643). Whether by the mechanism of breakage and rejoining or by the mechanism(s) of gene conversion, formation of heteroduplex DNA at homologously paired joints 5 can serve to transfer genetic sequence information from one DNA molecule to another. The ability of homologous recombination (gene conversion and classical strand breakage/rejoining) to transfer genetic sequence information between DNA molecules makes targeted homologous recombination a powerful method in genetic engineering and gene manipulation. The ability of mammalian and human cells to incorporate exogenous genetic material into genes 10 residing on chromosomes has demonstrated that these cells have the general enzymatic machinery for carrying out homologous recombination required between resident and introduced sequences. These targeted recombination events can be used to correct mutations at known sites, replace genes or gene segments with defective ones, or introduce foreign genes into cells. The efficiency of such gene targeting techniques is related to several parameters: the efficiency of DNA delivery into cells, 15 the type of DNA packaging (if any) and the size and conformation of the incoming DNA, the length and position of regions homologous to the target site (all these parameters also likely affect the ability of the incoming homologous DNA sequences to survive intracellular nuclease attack), the efficiency of recombination at particular chromosomal sites and whether recombinant events are homologous or nonhomologous. Over the past 10 years or so, several methods have been developed to introduce 20 DNA into mammalian cells: direct needle microinjection, transfection, electroporation, electroincorporation, retroviruses, adenoviruses, adeno-associated viruses; Herpes viruses, and other viral packaging and delivery systems, polyamidoamine dendimers, liposomes, and most recently techniques using DNA-coated microprojectiles delivered with a gene gun (called a biolistics device), or narrow-beam lasers (laser-poration). The processes associated with some types of gene transfer 25 have been shown to be both mutagenic and carcinogenic (Bardwell, (1989) Mutagenesis 4: 245), and these possibilities must be considered in choosing a transfection approach. The choice of a particular DNA transfection procedure depends upon its availability to the researcher, the technique's efficiency with the particular chosen target cell type, and the researchers concerns about the potential for generating unwanted genome mutations. For example, retroviral integration 30 requires dividing cells, always results in nonhomologous recombination events, and retroviral insertion within a coding sequence of nonhomologous (i.e., non-targeted) gene could cause cell mutation, by inactivating the gene's coding sequence (Friedmann, (1989) Science 244:1275). Newer retroviral based DNA delivery systems are being developed using defective retroviruses. However, these disabled viruses must be packaged using helper systems, are often obtained at low titer, and 3 WO 99/60108 PCT/US99/10731 recombination is still not site-specific, thus recombination between endogenous cellular retrovirus sequences and disabled virus sequences could still produce wild-type retrovirus capable of causing gene mutation. Adeno- or polyoma virus based delivery systems appear very promising (Samulski et al., (1991) EMBO J. 10: 2941; Gareis et al., (1991) Cell. Molec. Biol. 37: 191; Rosenfeld et al. (1992) 5 Cell 68: 143) although they still require specific cell membrane recognition and binding characteristics for target cell entry. Liposomes often show a narrow spectrum of cell specificities, and when DNA is coated externally on to them, the DNA is often sensitive to cellular nucleases. Newer polycationic lipospermines compounds exhibit broad cell ranges (Behr et al., (1989) Proc. NatI. Acad. Sci. USA 86: 6982) and DNA is coated by these compounds. In addition, a combination of neutral and cationic lipid 10 has been shown to be highly efficient at transfection of animal cells and showed a broad spectrum of effectiveness in a variety of cell lines (Rose et al., (1991) BioTechniques 10:520). Galactosylated bis acridine has also been described as a carrier for delivery of polynucleotides to liver cells (Haensler JL and Szoka FC (1992), Abstract V211 in J. Cell. Biochem. Supplement 16F, April 3-16, 1992, incorporated herein by reference). Electroporation also appears to be applicable to most cell types. 15 The efficiency of this procedure for a specific gene is variable and can range from about one event per 3 x 10' transfected cells (Thomas and Capecchi, (1987) Cel5_: 503) to between one in 107 and 108 cells receiving the exogenous DNA (Koller and Smithies, (1989) Proc. Natl. Acad. Sci. (U.S.A.) : 8932). Microinjection of exogenous DNA into the nucleus has been reported to result in a high frequency of stable transfected cells. Zimmer and Gruss (Zimmer and Gruss (1989) Nature 338: 150) 20 have reported that for the mouse hox1.1 gene, 1 per 150 microinjected cells showed a stable homologous site specific alteration. Several methods have been developed to detect and/or select for targeted site-specific recombinants between vector DNA and the target homologous chromosomal sequence (see, Capecchi, (1989) Science 244: 1288 for review). Cells which exhibit a specific phenotype after site-specific 25 recombination, such as occurs with alteration of the hprt gene, can be obtained by direct selection on the appropriate growth medium. Alternatively, a selective marker sequence such as neo can be incorporated into a vector under promoter control, and successful transfection can be scored by selecting G418r cells followed by PCR to determine whether neo is at the targeted site (Joyner et al., (1989) Nature 3: 153). A positive-negative selection (PNS) procedure using both neo and HSV-tk 30 genes allows selection for transfectants and against nonhomologous recombination events, and significantly enriched for desired disruption events at several different mouse genes (Mansour et al., (1988) Nature 336: 348). This procedure has the advantage that the method does not require that the targeted gene be transcribed. If the targeted gene is transcribed, a promoter-less marker gene can be incorporated into the targeting construct so that the gene becomes activated after homologous 35 recombination with the target site (Jasin and Berg, (1988) Genes and Development 2: 1353; Doetschman et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 8583; Dorini et al., (1989) Science 243: 4 WO 99/60108 PCT/US99/10731 1357; Itzhaki and Porter, (1991) Nucl. Acids Res. 19: 3835). Recombinant products produced using vectors with selectable markers often continue to retain these markers as foreign genetic material at the site of transfection, although loss does occur. Valancius and Smithies (Valancius and Smithies, (1991) Molec. Cellular Biol. 11: 1402) have described an "in-out" targeting procedure that allowed a 5 subtle 4-bp insertion modification of a mouse hprt target gene. The resulting transfectant contained only the desired modified gene sequence and no selectable marker remained after the "out" recombination step. Cotransformation of cells with two different vectors, one vector contained a selectable gene and the other used for gene disruption, increases the efficiency of isolating a specific targeting reaction (Reid et al., (1991) Molec. Cellular Biol. 11: 2769) among selected cells that are 10 subsequently scored for stable recombinants. Unfortunately, exogenous sequences transferred into eukaryotic cells undergo homologous recombination with homologous endogenous sequences only at very low frequencies, and are so inefficiently recombined that large numbers of cells must be transfected, selected, and screened in order to generate a desired correctly targeted homologous recombinant (Kucherlapati et al. (1984) 15 Proc. NatI. Acad. Sci. (U.S.A.) 81: 3153; Smithies, 0. (1985) Nature .1Z: 230; Song et al. (1987) Proc. NatI. Acad. Sci. (U.S.A.) 84: 6820; Doetschman et al. (1987) Nature 330: 576; Kim and Smithies (1988) Nucleic Acids Res. 16: 8887; Doetschman et al. (1988) op.cit.; Koller and Smithies (1989) op.cit.; Shesely et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) B8: 4294; Kim et al. (1991) Gene 103: 227, which are incorporated herein by reference). 20 Koller et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.), B: 10730 and Snouwaert et al. (1992) Science 257: 1083, have described targeting of the mouse cystic fibrosis transmembrane regulator (CFTR) gene for the purpose of inactivating, rather than correcting, a murine CFTR allele. Koller et al. employed a large (7.8kb) homology region in the double-stranded DNA targeting construct, but nonetheless reported a low frequency for correct targeting (only 1 of 2500 G418-resistant cells were correctly 25 targeted). Thus, even targeting constructs having lone homology regions are inefficiently targeted. Several proteins or purified extracts having the property of promoting homologous recombination (i.e., recombinase activity) have been identified in prokaryotes and eukaryotes (Cox and Lehman (1987) Ann. Rev. Biochem. 56: 229; Radding, C.M. (1982) op.cit.; Madiraju et al. (1988) Proc. Nati. Acad. Sci. (U.S.A.) 85: 6592; McCarthy et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 5854; Lopez et al. (1987) 30 op.cit., which are incorporated herein by reference). These general recombinases presumably promote one or more steps in the formation of homologously-paired intermediates, strand-exchange, gene conversion, and/or other steps in the process of homologous recombination. 5 WO 99/60108 PCT/US99/10731 The frequency of homologous recombination in prokaryotes is significantly enhanced by the presence of recombinase activities. Several purified proteins catalyze homologous pairing and/or strand exchange in vitro, including: E. coli recA protein, the T4 uvsX protein, and the rec protein from Ustilago maydis. Recombinases, like the recA protein of E. coli are proteins which promote strand 5 pairing and exchange. The most studied recombinase to date has been the recA recombinase of E. coli, which is involved in homology search and strand exchange reactions (see, Cox and Lehman (1987) op.cit.). RecA is required for induction of the SOS repair response, DNA repair, and efficient genetic recombination in E. coli. RecA can catalyze homologous pairing of a linear duplex DNA and a homologous single strand DNA in vitro. In contrast to site-specific recombinases, proteins like recA 10 which are involved in general recombination recognize and promote pairing of DNA structures on the basis of shared homology, as has been shown by several in vitro experiments (Hsieh and Camerini Otero (1989) J. Biol. Chem. 264: 5089; Howard-Flanders et al. (1984) Nature 309: 215; Stasiak et al. (1984) Cold Spring Harbor Symp. Quant. Biol. 49: 561; Register et al. (1987) J. Biol. Chem. 262: 12812). Several investigators have used recA protein in itro to promote homologously paired triplex 15 DNA (Cheng et al. (1988) J. Biol. Chem. 263: 15110; Ferrin and Camerini-Otero (1991) Science 354: 1494; Ramdas et al. (1989) J. Biol Chem. 264:11395; Strobel et al. (1991) Science 254: 1639; Hsieh et al. (1990) op.cit.; Rigas et al. (1986) Proc. Natl. Acad. Sci. (U.S.A.) 3: 9591; and Camerini-Otero et al. U.S. 7,611,268 (available from Derwent), which are incorporated herein by reference). Unfortunately many important genetic engineering manipulations involving homologous recombination, 20 such as using homologous recombination to alter endogenous DNA sequences in a living cell, cannot be done in vitro. Further, gene therapy requires highly efficient homologous recombination of targeting vectors with predetermined endogenous target sequences, since selectable marker selection schemes, such as those currently available in the art, are not usually practicable in human beings. Thus, there exists a need in the art for methods of efficiently altering predetermined endogenous 25 genetic sequences by homologous pairing and homologous recombination in vivo by introducing one or more exogenous targeting polynucleotide(s) that efficiently and specifically homologously pair with a predetermined endogenous DNA sequence. There exists a need in the art for high-efficiency gene targeting, so as to avoid complex in vitro selection protocols (e.g., neo gene selection with G418), which are of limited utility for in vyv gene therapy on affected individuals 30 SUMMARY OF THE INVENTION It is an object of the present invention to provide methods for targeting an exogenous polynucleotide to a predetermined endogenous DNA target sequence in a target cell with high efficiency and with sequence specificity. Exogenous polynucleotides, are localized (or targeted) to one or more predetermined DNA target sequence(s) by homologous pairing in viyv. Such targeted homologous 35 pairing of exogenous polynucleotides to endogenous DNA sequences in vivo may be used: (1) to 6 WO 99/60108 PCT/US99/10731 target chemical substituents in a sequence-specific manner in vjv, (2) to correct or to generate genetic mutations in endogenous DNA sequences by homologous recombination and/or gene conversion, (3) to produce homologously targeted transgenic animals and plants at high efficiency, and (4) in other applications (e.g., targeted drug delivery) based on in vivo homologous pairing. Some 5 embodiments of the invention employ targeted exogenous polynucleotides to correct endogenous mutant gene alleles in human cells; the invention provides methods and compositions for correcting disease alleles involved in producing human genetic diseases, such as inherited genetic diseases (e.g., cystic fibrosis) and neoplasia (e.g., neoplasms induced by somatic mutation of an oncogene or tumor suppressor gene, such as p53, or viral genes associated with neoplasia, such as HBV genes). 10 In one embodiment, at least one exogenous polynucleotide is targeted to a predetermined endogenous DNA sequence and alters the endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination within and/or flanking the predetermined endogenous DNA sequence. Generally, two complementary exogenous polynucleotides are used for targeting an endogenous DNA sequence. Typically, the targeting polynucleotide(s) are introduced 15 simultaneously or contemporaneously with one or more recombinase species. Alternatively, one or more recombinase species may be induced or produced in vivo, for example by expression of a heterologous expression cassette in a cell containing the preselected target DNA sequence. It is another object of the invention to provide methods whereby at least one exogenous polynucleotide containing a chemical substituent can be targeted to a predetermined endogenous DNA sequence in a 20 metabolically-active or intact living target cell, permitting sequence-specific targeting of chemical substituents such as, for example cross-linking agents, metal chelates (e.g., iron/EDTA chelate for iron catalyzed cleavage), topoisomerases, endonucleases, exonucleases, ligases, phosphodiesterases, photodynamic porphyrins, free-radical generating drugs, chemotherapeutic drugs (e.g., adriamycin, doxirubicin), intercalating agents, base-modification agents, immunoglobulin chains, oligonucleotides, 25 and other substituents. The methods of the invention can be used to target such a chemical substituent to a predetermined DNA sequence by homologous pairing for various applications, for example: producing sequence-specific strand scission(s), producing sequence-specific chemical modifications (e.g., base methylation, strand cross-linking), producing sequence-specific localization of polypeptides (e.g., topoisomerases, helicases, proteases), producing sequence-spetcific localization of 30 polynucleotides (e.g., loading sites for transcription factors and/or RNA polymerase), and other applications. It is another object of the present invention to provide methods for correcting a genetic mutation in an endogenous DNA target sequence, such as a sequence encoding an RNA or a protein. For example, the invention can be used to correct genetic mutations, such as base substitutions, additions, and/or 7 WO 99/60108 PCT/US99/10731 deletions, by converting a mutant DNA sequence that encodes a non-functional, dysfunctional, and/or truncated polypeptide into a corrected DNA sequence that encodes a functional polypeptide (e.g., has a biological activity such as an enzymatic activity, hormone function, or other biological property). The methods and compositions of the invention may also be used to correct genetic mutations or 5 dysfunctional alleles with genetic lesions in non-coding sequences (e.g., promoters, enhancers, silencers, origins of replication, splicing signals). In contradistinction, the invention also can be used to target DNA sequences for inactivating gene expression; a targeting polynucleotide can be employed to make a targeted base substitution, addition, and/or deletion in a structural or regulatory endogenous DNA sequence to alter expression of one or more genes, typically by knocking out at least one allele 10 of a gene (i.e., making a mutant, nonfunctional allele). The invention can also be used to correct disease alleles, such as a human or non-human animal CFTR gene allele associated with cystic fibrosis, by producing a targeted alteration in the disease allele to correct a disease-causing lesion (e.g., a deletion). It is a further object of the invention to provide methods and compositions for high-efficiency gene 15 targeting of human genetic disease alleles, such as a CFTR allele associated with cystic fibrosis or an LDL receptor allele associated with familial hypercholesterolemia. In one aspect of the invention, targeting polynucleotides having at least one associated recombinase are targeted to cells in vivo (i.e., in an intact animal) by exploiting the advantages of a receptor-mediated uptake mechanism, such as an asialoglycoprotein receptor-mediated uptake process. In this variation, a targeting polynucleotide is 20 associated with a recombinase and a cell-uptake component which enhances the uptake of the targeting polynucleotide- recombinase into cells of at least one cell type in an intact individual. For example, but not limitation, a cell-uptake component typically consists of: (1) a galactose-terminal (asialo-) glycoprotein (e.g., asialoorosomucoid) capable of being recognized and internalized by specialized receptors (asialoglycoprotein receptors) on hepatocytes in viv, and (2) a polycation, such 25 as poly-L-lysine, which binds to the targeting polynucleotide, usually by electrostatic interaction. Typically, the targeting polynucleotide is coated with recombinase and cell-uptake component simultaneously so that both recombinase and cell-uptake component bind to the targeting polynucleotide; alternatively, a targeting polynucleotide can be coated with recombinase prior to incubation with a cell-uptake component; alternatively the targeting polynucleotide can be coated with 30 the cell-uptake component and introduced into cells contemporaneously with a separately delivered recombinase (e.g., by targeted liposomes containing one or more recombinase). The invention also provides methods and compositions for diagnosis, treatment and prophylaxis of genetic diseases of animals, particularly mammals, wherein a recombinase and a targeting polynucleotide are used to produce a targeted sequence modification in a disease allele of an 35 endogenous gene. The invention may also be used to produce targeted sequence modification(s) in a 8 WO 99/60108 PCT/US99/10731 non-human animal, particularly a non-human mammal such as a mouse, which create(s) a disease allele in a non-human animal. Sequence-modified non-human animals harboring such a disease allele may provide useful models of human and veterinary disease(s). Alternatively, the methods and compositions of the invention can be used to provide nonhuman animals having homologously 5 targeted human disease alleles integrated into a non-human genome; such non-human animals may provide useful experimental models of human or other animal genetic disease, including neoplastic and other pathogenic diseases. It is also an object of the invention to provide methods and compositions for recombinase- enhanced positioning of a targeting polynucleotide to a homologous sequence in an endogenous chromosome to 10 form a stable multistrand complex, and thereby alter expression of a predetermined gene sequence by interfering with transcription of sequence(s) adjacent to the multistrand complex. Recombinase(s) are used to ensure correct homologous pairing and formation of a stable multistrand complex, which may include a double-D loop structure. For example, a targeting polynucleotide coated with a recombinase may homologously pair with an endogenous chromosomal sequence in a structural or regulatory 15 sequence of a gene and form a stable multistrand complex which may: (1) constitute a significant physical or chemical obstacle to formation of or procession of an active transcriptional complex comprising at least an RNA polymerase, or (2) alter the local chromatin structure so as to alter the transcription rate of gene sequences within about 1 to 500 kilobases of the multistrand complex. It is another object of the invention to provide methods and compositions for treating or preventing 20 acquired human and animal diseases, particularly parasitic or viral diseases, such as human hepatitis B virus (HBV) hepatitis, by targeting viral gene sequences with a recombinase-associated targeting polynucleotide and thereby inactivating said viral gene sequences and inhibiting viral-induced pathology. It is a further object of the invention to provide compositions that contain exogenous targeting 25 polynucleotides, complementary pairs of targeting polynucleotides, chemical substituents of such polynucleotides, and recombinase proteins used in the methods of the invention. Such compositions may include a targeting or cell-uptake components to facilitate intracellular uptake of a targeting polynucleotide, especially for in yj gene therapy and gene modification. In accordance with the above objects, the present invention provides methods for targeting and 30 altering, by homologous recombination, a pre-selected target nucleic acid sequence in a cell to make a targeted sequence modification. The methods comprise introducing into at least one cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially 9 WO 99/60108 PCT/US99/10731 complementary to each other and comprise a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence. In an additional aspect, the invention provides compositions for producing targeted modifications of target sequences, including disease alleles, comprising two substantially complementary 5 single-stranded targeting polynucleotides and at least one recombinase. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Homologous targeting of recA-coated chromosome 1 alpha-satellite polynucleotides in metabolically active cell nuclei. The homologously targeted biotinylated polynucleotides were visualized by addition of FITC-avidin followed by washing to remove unbound FITC. Signals were 10 visualized using a Zeiss Confocal Laser Scanning Microscope (CLSM-10) with 488 nm argon laser beam illumination for FITC-DNA detection. Top left -localized FITC-DNA signals in the cell nucleus. Lower left -enhanced image of FITC-DNA signals in the cell nucleus. Upper right - image of FITC DNA signals overlaid on the phase image of nucleus. Lower right - phase image of the center of the cell nucleus showing nucleoli. Note: all images except lower right were photographed at the same 15 focus level (focus unchanged between these photos). Figs. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2J, 2K, and 2L. RecA protein-mediated native FISH in metabolically active cell nuclei. Hep-2 cell nuclei from cells encapsulated in agarose were incubated with RecA-coated biotinylated p53 DNA (A-I) or RecA-coated biotinylated chromosome 1 satellite Ill DNA probes (K-L). Panels B-I show FISH signals in digital images from serial CLSM optical sections 20 of FITC-labeled p53 probe DNA incubated in metabolically active Hep-2 nuclei. The phase image of a representative nucleous in shown in Panel A and was sectioned by CLSM. Digital images in Panels B-H were serially overlaid upon one another to produce the composite digital image shown in Panel I containing all three FITC labeled p53 FISH signals. The effect of cssDNA probe concentration and RecA protein on efficiency of native dsDNA hybridization in metabolically active nuclei is shown in 25 Panel J. The percentage of labeled RecA coated or uncoated p53 cssDNA is shown as a function of the amount of p53 DNA probe per hybridization reaction. Closed circles show hybridization reactions with RecA-coated p53 cssDNA probe, open triangles show control reactions without RecA protein coating of p53 cssDNA probe. Panel K shows the FISH digital image in Panel L overlaid onto the phase image. 30 Fig. 3. Genetic map of mammalian expression lacZ plasmid pMC1 lacXpA with an 11 base insertion in the Xba linker site. 10 WO 99/60108 PCT/US99/10731 Fig. 4. Genetic map of the mammalian expression lacZ plasmid pMC11acpA, with an insertion mutation. Fig. 5. PCR products and primers from the lacZ (B-galactosidase) gene sequence. The location of the 11 bp Xba linker is shown. 5 Fig. 6. Tests for alteration of an insertion mutation in the lacZ gene of a eukaryotic expression vector. NIH 3T3 cells were needle microinjected with five types of plasmids: Two plasmids contained a wild-type R-galactosidase gene (pMC1 lacpa or pSV-B-gal [Promega]); a plasmid with a mutant B-gal gene (pMC1lacXpa); pMC1lacXpa plasmid incubated with a protein uncoated wild-type 276-mer DNA; or pMC1 lacXpa plasmid reacted and D-looped with RecA-coated wild-type 276-mer DNA. The 10 wild-type 276-mer DNA was heat denatured and either coated or not coated with RecA protein in a standard RecA protein coating reaction protocol (Sena and Zarling, supra). Following a 10-min RecA coating reaction, the RecA-coated complementary single-stranded 276-mers were incubated at 37 0 C for 60 min. with the mutant target plasmid to allow hybrid formation. A 60 min incubation of the mutant target plasmid DNA with uncoated complementary single-stranded normal wild-type 276-mers was 15 carried out as a control and hybrids were not formed. The B-galactosidase activity in needle microinjected cells using the wild-type plasmids is shown for comparison. On average, about 50% of the total microinjected cells survived. The numbers of surviving cells scoring blue with the mutant plasmid hybridized with RecA-coated CSS DNA and reacted with non-RecA-coated CSS DNA samples (3, 4 and 5) were compared with fourfold P 2 tests. The frequency of corrected blue cells in 20 the RecA-coated CSS DNA samples (Sample 5; 6 out of 168) is significantly higher than that of either Sample 3 or Sample 4. The frequency of corrected RecA-coated CSS DNA probe:target hybrids blue cells in Sample 5 is significantly higher than that of Sample 4 at the 5% significance level (P 2 = 3.76 >

P

2 005 ). The frequency of corrected blue cells in Sample 5 containing RecA-coated CSS DNA probe:target hybrids is significantly higher than that of Sample 3 at the 1% significance level (P 2 = 6.28 25 > P 2 0 e,). When Samples 3 and 4 are combined and compared with Sample 5, the frequency of corrected blue cells in Sample 5 is significantly higher than that of the combined sample at the 0.1% signficance level (P 2 = 9.9 > p 2 0 . Fig. 7A. Southern hybridization analysis of the 687-bp fragment amplified from genomic DNA. Electrophoretic migration of a 687-bp DNA fragment generated with primers CF1 and CF6 from 30 genomic DNA of 3CFTE29o-cells which were capillary needle-microinjected with the 491-nucleotide DNA fragment in the presence of recA protein (lane 2) or transfected as a protein-DNA-lipid complex where the 491-nucleotide fragments were coated with recA protein (+; lane 3). The control DNA was amplified from nontransfected 3CFTE29o-cultures (lane 1). 11 WO 99/60108 PCT/US99/10731 Fig. 7B. Autoradiographic analysis of DNA transferred to Gene Screen Plus filters and hybridized with a 32 P-labeled oligonucleotide specific for normal exon 10 sequences in the region of the AF508 mutation. Cells transfected by micro-injection or protein-lipid-DNA complexes both were positive for homologous targeting, whereas control cells were not. 5 Fig. 8A. Analysis of DNA from cells electroporated or transfected with CSS DNA encapsulated in a protein-lipid complex. Allele-specific PCR amplification of the 687/684 bp DNA fragment amplified in the first round with primers CF1 and oligo N (N) or oligo AF (AF). Ethidium bromide-stained 300 bp. DNA fragment separated by electrophoresis in a 1% agarose gel. The DNA in each lane is as follows: lane 1, 100-bp marker DNA; lane 2, control 16HBE14o-cell DNA amplified with the CF1/N primer pair; 10 lane 3, nontransfected ECFTE29o-cell DNA amplified with CF1/N primers; lane 4, nontransfected ECFTE29o-cell DNA amplified with CF1I/AF primers; lane 5, DNA from ECFTE29o-cells electroporated with recA-coated 491-nucleotide fragments and amplified with CF1/N primers; lane 6, DNA from ECFTE29o-cells transfected with recA-coated 491-nucleotide fragment encapsulated in a protein-lipid complex and amplified with CF1/N primers. 15 Fig. 8B. Autoradiographic analysis of the DNA in Fig. IIA transferred to Gene Screen Plus filters and hybridized with 1 2 P-labeled oligo N probe. Samples in lanes 1-5 for the autoradiographic analysis are equivalent to samples in lanes 2-6 in Fig. IIA. Fig. 9. PCR analysis of 3CFTE29o-genomic DNA reconstructed with the addition of 2 x 105 copies of recA-coated 491-nucleotide CSS DNA fragments per microgram of genomic DNA. This number of 20 CSS DNA fragments represents the total number of DNA copies microinjected into cells and tests whether the 491-nucleotide fragment can act as a primer for the 687/684-bp fragment amplification. DNA was amplified as described in Fig. 8A. When the second round of amplification was conducted with CF1 and oligo N primers (lane 2), the 300-bp DNA band was not detected when aliquots of the amplification reaction were separated electrophoretically. Amplification of the ECFTE29o/491 bp DNA 25 fragment with the CF1/oligo AF primer pair produced a 299-bp DNA product (lane 1). Marker DNA is in lane 3. Figure 10 depicts the scheme for the recombination assay used in Example 4. . Fig. 11 shows RecA mediated cssDNA targeting to dsDNA with deletions produces a mixed population of probe:target hybrids. The biotinylated cssDNA probes were denatured and coated with RecA at 30 370C as described in Material . The reaction mixture was incubated for 60 minutes at 37'C. All reactions were stopped by deproteinization with 1.2% SDS and separated by electrophoresis on a 20 cm X 25 cm 1% agarose gel. The gel was run overnight at 30V then blotted onto a positively charged 12 WO 99/60108 PCT/US99/10731 Tropilon Plus (TROPIX) membrane. The DNA was monitored for the presence of unhybridized probe or probe:target hybrids using an alkaline phosphatase based chemiluminescent detection of biotin. When the membranes were exposed to X-ray film and developed, it is evident that cssDNA probes will hybridize to dsDNA targets which are completely homologous, as well as dsDNA targets which contain 5 a deletion (lanes 3 and 6, respectively). RecA mediated cssDNA targeting to completely homologous dsDNA (pRD.0) forms a probe:target hybrid whose electrophoretic mobility is comparable to the electrophoretic mobility of completely relaxed Form I DNA, which is similar to the mobility of Form I DNA (lanes 3, 8, and 10), referred to as the rl* hybrid. RecA mediated hybridization of cssDNA to dsDNA containing a 59 base pair deletion (pRD.59), a probe:target hybrid that migrates to a position 10 similar to Form I DNA (lane 6), is referred to as the 1* hybrid. Fig. 12 shows data for the enhanced homologous recombination (EHR) of cssDNA probe:target hybrids in E. coli, as per Example 4. The homologously targeted probe:target hybrids have enhanced homologous recombination frequencies in recombination proficient cells. cssDNA probe:target hybrids were formed as described in the legend of Figure 11 and were introduced into RecA+ and RecA-E. coli 15 as in described Figure 12. The molar ratio of cssDNA probe:target in the in vitro targeting reaction varied from 1:1 to 1:5.6. The % recombinant/total colonies is the percentage of blue colonies in the total population of ampicillin-resistant colonies. Groups with 0% recombinants did not produce any blue colonies in at least 105 plated colonies. Plasmid DNA was isolated from blue colonies that were serially propagated for three generations to determine if homologous recombination stably occurred in 20 the lacZ gene. Fig. 13 shows double D-loop hybrids with internal homology clamps. A) Duplex target DNA (thin line) is completely homologous to the cssDNA probe (thick) and each probe strand can pair with its complementary strand in the target. B) Duplex target has a deletion with respect to the cssDNA probe. The deleted region is indicated with a dashed line. The region of the cssDNA probes homologous to 25 the deleted region in the target can re-pair with each other forming a stable hybrid complex. C) Duplex target has an insertion (dashed line) with respect to the cssDNA probe. Structures on the left show the re-annealing of cssDNA probe or target strands to form internal homology clamps. Structures on the right show the presence of unpaired regions in comparable single D-loop hybrids. Figs. 14A and 14B. Figure 14A depicts the Maps of Plasmids pRD.0 and pRD.59. Relative positions 30 of cssDNA probes IP290 and CP443, PCR primers 1A and 4B, restriction endonuclease sites EcoRI, Scal, and Dral are indicated. The alpha peptide sequence of the LacZ gene is indicated. Note the deletion ()) in pRD.59 is approximately equidistant from the ends of primers 1A and 4B. Figure 14B). Time course for cssDNA probe:target hybrid formation with linear dsDNA targets. Biotinylated, RecA coated cssDNA probe IP290 was hybridized as described to Scal-digested plasmids pRD.0 and 13 WO 99/60108 PCT/US99/10731 pRD.59 carrying 0 or 59 bp deletion, respectively at the EcoR1 site in pRD.O. Probe IP290 is completely homologous to pRD.0, but has a 59 bp insertion with respect to pRD.59. Fig. 15 depicts the formation of cssDNA probe:target hybrids formed with linear dsDNA targets containing small deletions. A) Plasmid constructs and probes used in this study. A series of plasmids 5 with defined deletions were constructed from the EcoR1 site of pRD.0 (pbluescriptllSK+ (Stratagene) as described in Example 5. Each plasmid is named for the size of the deletion, as indicated on the left. A series of cssDNA probes were labelled and constructed by PCR from various primers which flank the deleted region. Probes were made from either pRD.0 or the deleted plasmids and named for the size of the probe when made from pRD.0 (2960 bp). For example, p527 is 527 bp long. When the 10 cssDNA probes are produced from pRD.0 and targeted to plasmids containing deletions, the probe is called IP527 to indicate that the insertion probe (IP) has an insertion with respect to the target. When the probe is made from one of the targets with a deletion and then, targeted to pRD.0, the probe is called DP527 to indicate that the deletion probe (DP) has a deletion with respect to pRD.O. Control probe CP443 is made from a region of pRD.0 that does not contain any insertions or deletions. The 15 limits of the deleted regions in the plasmid DNA target are indicated by dashed lines and the size limits of cssDNA probes are indicated by solid lines. B) Biotinylated cssDNA probes IP527, IP407, and CP443 were coated with RecA protein and hybridized at 37*C to a series of linear duplex DNA targets containing deletions ranging in size from 0 to 447 bp. The products of the targeting reaction were deproteinized and separated on a 1 % TAE-agarose gel and then transferred to nylon 20 membranes as described in Example 5. Biotinylated DNA was detected with a chemiluminescent substrate as described. The extent of hybrid product formation of Form Ill DNA targets was determined by densitometry of the autoradiographs. The relative amount of hybrid formed between RecA coated cssDNA probes IP527 and IP407 is shown in (B). Error bars are indicated. The amount of probe:target hybrids formed with each target DNA was normalized by the amount of probe:target 25 hybrids formed with control probe CP443 which hybridizes to the target located in a region which is a significant distance away from the deletion site. Examples of the cssDNA probe:target hybrid formed with linear targets are shown in the autoradiogram (C). In Fig. 15(D) the difference in the percent hybrid formation between cssDNA probes IP527 and IP407 are plotted from the data shown in (B). Fig. 16 depicts that insertions and deletions have the same effect on the relative efficiency of 30 probe:target hybrid formation. RecA-coated cssDNA probes IP215 made from pRD.0 was targeted to Scal-digests of plasmids pRD.0, pRD.8, pRD.25, and pRD.59 and compared to similar reactions of DP215 cssDNA probes made from pRD.0, pRD.8, pRD.25, and pRD.59 and targeted to pRD.O. The effect of insertions in the cssDNA probe (dark line) is compared with deletions in the cssDNA probe (shaded line) of the same size. The relative level of hybrid formation for each cssDNA probe with a 14 WO 99/60108 PCT/US99/10731 heterologous target is normalized by the level of hybridization with the homologous target, respectively. The data represents an average of three experiments. Error bars are indicated. Figs 17A, 17B and 17C. Figure 17A depict the formation of stable double-D-Loop hybrids in linear dsDNA targets containing large deletions. Biotinylated cssDNA probe IP1246 was coated with RecA 5 protein and targeted to Scal digests of the indicated plasmids as described herein. The relative amount of hybrid formation formed between RecA-coated cssDNA probes and plasmids with deletions ranging from 0-967 bp was normalized to the amount of probe:target hybrids formed with control probe CP443. Autoradiograph (17A) shows the biotinylated cssDNA probes or probe:target hybrids. The position of the untargeted Scal-digested (Formill) marker for each of the plasmids are indicated on the 10 right. The relative level of hybrid formation (B) of each of the bands in (A) was normalized to the level of hybrid formation with control cssDNA probe CP443, as described herein. The relative position of the cssDNA probes with respect to the position of the deletion in the target DNA is shown in (C). Figs. 18A, 18B, 18C and 18D depict the formation of restriction endonuclease sites in probe:target hybrids. The probe:target hybrids formed between probe IP290 and pRD.0 and pRD.59 targets were 15 deproteinized by extraction with chloroform:phenol:isoamyl alcohol and chloroform. Restriction enzyme treated DNA samples were incubated with EcoRI for three hours before separation on a 1% agarose gel and transferred onto a nylon membrane. The ethidium bromide stained DNA of the products of the targeting reactions formed between cssDNA probe IP290 and circular plasmid targets pRD.0 or pRD.59 (A and B) and autoradiographs showing the positions of biotinylated cssDNA 20 probe:target hybrids (C and D) are shown. The positions of form I and form Ill markers of pRD.0 are shown on the right. The positions of the pRD59 hybrids I* (form 1) and rl* (relaxed) are shown on the left. Fig. 19 depicts the thermal stability of relaxed and non-relaxed probe:target hybrids. The RecA mediated cssDNA targeting reaction was performed with the cssDNA probe IP290 and the dsDNA 25 target pRD.59, as described herein. The probe:target hybrids were deproteinized with 1.2% SDS and then incubated for 5 minutes at the indicated temperatures. The thermally melted products were then separated on a 1% agarose gel and blotted onto a positively charged Tropilon membrane. Autoradiograph shows the position of biotinylated cssDNA probe:target hybrids I* (forml) and rl* (relaxed) as shown on the left. 30 Figs. 20A and 20B. The organization of the mouse OTC gene. Sequence of cssDNA probes and PCR primers used in this study are indicated. Sizes of the exons in base pairs are indicated. The relative position of PCR primers M9, M8 and M1 1 are shown. B) Map of plasmid pTAOTC1. A 250 bp 15 WO 99/60108 PCT/US99/10731 fragment containing the normal OTC exon 4 sequence and surrounding introns were cloned into the EcoRV site of pbluescript SK (+) (Stratagene). Fig. 21. Sequence analysis of exon 4 of the mouse OTC gene in founder mice. PCR amplification of genomic DNA from tail biopsies of a pool of all of the homozygous (spf-ash/spf-ash) females used as 5 egg donors and each indicated individual founder mice were sequenced using cycle sequencing with the M1 1 primer (Cyclist kit, Stratagene). The DNA sequence surrounding the spf-ash locus (arrow) in the OTC gene is shown. Fig. 22. Germline transmission of OTC+ allele corrected by EHR. The inheritance patterns of the OTC alleles are depicted. Legend indicates the genotype and/or phenotype of the FO, F1, and F2 10 mice produced from microinjected zygotes obtained from the cross of homozygous (spf-ash/spf-ash) mutant females and normal males (top). The genotype of FO and F1 animals were determined by DNA sequencing and the typing of F2 animals as deduced by phenotype. Control cross A of (hemizygous spf-ash/Y) mutant FO male with normal (+/+) females and control cross B of heterozygous (spf-ash/+) F1 females with a normal male are indicated. The number below the boxes 15 or circles indicate the total number of mice of each type produced from each cross. Total numbers of mice counted are representative of 2-4 litters. Mouse #213 and #1014 (noted by arrow) are F1 animals that carry a germline transmitted gene corrected allele from mosaic HR gene corrected male mouse #16. Fig. 23. Germline transmission of corrected allele of FO male #16. Pictures of F1 progeny from the 20 cross of mouse #16 with homozygous (spf-ash/spf-ash) females (top). This cross produced several pups with spf-ash mutant phenotypes (middle) and one F1 pup (#1014) with a normal phenotype. Three views of mouse #1014 are shown (bottom). All of the F1 animals were two weeks old at the time of photography. DEFINITIONS 25 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below. 30 As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage (immunology -A Synthesis, 2nd Edition, E.S. Golub and D.R. Green, Eds., Sinauer Associates, Sunderland, Massachusetts (1991), which is incorporated herein by reference). 16 WO 99/60108 PCTIUS99/10731 By Anucleic acid@, Aoligonucleotide@, and Apolynucleotide@ or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., 5 Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate, phosphorodithioate, 0-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University 10 Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). These modifications of the ribose-phosphate backbone or bases may be done to facilitate the addition of other moieties such as chemical constituents, including 2' O-methyl and 5' modified substituents, as discussed below, or to 15 increase the stability and half-life of such molecules in physiological environments. The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo-and ribo nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, 20 inosine, xathanine and hypoxathanine, etc. Thus, for example, chimeric DNA-RNA molecules may be used such as described in Cole-Strauss et al., Science 273:1386 (1996) and Yoon et al., PNAS USA 93:2071 (1996), both of which are hereby incorporated by reference. In general, the targeting polynucleotides may comprise any number of structures, as long as the changes do not substantially effect the functional ability of the targeting polynucleotide to result in 25 homologous recombination. For example, recombinase coating of alternate structures should still be able to occur. As used herein, the terms Apredetermined endogenous DNA sequence" and "predetermined target sequence" refer to polynucleotide sequences contained in a target cell. Such sequences include, for example, chromosomal sequences (e.g., structural genes, regulatory sequences including promoters 30 and enhancers, recombinatorial hotspots, repeat sequences, integrated proviral sequences, hairpins, palindromes), episomal or extrachromosomal sequences (e.g., replicable plasmids or viral or parasitic replication intermediates) including chloroplast and mitochondrial DNA sequences. By "predetermined" or Apre-selected@ it is meant that the target sequence may be selected at the discretion of the practitioner on the basis of known or predicted sequence information, and is not 17 WO 99/60108 PCT/US99/10731 constrained to specific sites recognized by certain site-specific recombinases (e.g., FLP recombinase or CRE recombinase). In some embodiments, the predetermined endogenous DNA target sequence will be other than a naturally occurring germline DNA sequence (e.g., a transgene, parasitic, mycoplasmal or viral sequence). An exogenous polynucleotide is a polynucleotide which is 5 transferred into a target cell but which has not been replicated in that host cell; for example, a virus genome polynucleotide that enters a cell by fusion of a virion to the cell is an exogenous polynucleotide, however, replicated copies of the viral polynucleotide subsequently made in the infected cell are endogenous sequences (and may, for example, become integrated into a cell chromosome). Similarly, transgenes which are microinjected or transfected into a cell are exogenous 10 polynucleotides, however integrated and replicated copies of the transgene(s) are endogenous sequences. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., may be similar or identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide 15 sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. As outlined below, preferably, the homology is at least 50-70%, preferably 85%, and more preferably 95% identical. Thus, the complementarity between two single-stranded targeting polynucleotides need not be perfect. For illustration, the nucleotide sequence "TATAC" corresponds to a reference 20 sequence "TATAC@ and is perfectly complementary to a reference sequence "GTATA". The terms "substantially corresponds to" or "substantial identity" or Ahomologous@ as used herein denotes a characteristic of a nucleic acid sequence, wherein a nucleic acid sequence has at least about 60 percent sequence identity as compared to a reference sequence, typically at least about 75 percent sequence identity, and preferably at least about 95 percent sequence identity as compared to 25 a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, the reference sequence is at least 12-18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long. 30 ASubstantially complementary" as used herein refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence. In general, targeting efficiency increases with the length of the targeting polynucleotide portion that is substantially complementary to a reference sequence present in the target DNA. 18 WO 99/60108 PCT/US99/10731 "Specific hybridization@ is defined herein as the formation of hybrids between a targeting polynucleotide (e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions as compared to the predetermined target DNA sequence) and a predetermined target DNA, wherein the targeting polynucleotide preferentially hybridizes to the predetermined target DNA such 5 that, for example, at least one discrete band can be identified on a Southern blot of DNA prepared from target cells that contain the target DNA sequence, and/or a targeting polynucleotide in an intact nucleus localizes to a discrete chromosomal location characteristic of a unique or repetitive sequence. In some instances, a target sequence may be present in more than one target polynucleotide species (e.g., a particular target sequence may occur in multiple members of a gene family or in a known 10 repetitive sequence). It is evident that optimal hybridization conditions will vary depending upon the sequence composition and length(s) of the targeting polynucleotide(s) and target(s), and the experimental method selected by the practitioner. Various guidelines may be used to select appropriate hybridization conditions (see, Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology, Volume 15 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA., which are incorporated herein by reference. Methods for hybridizing a targeting polynucleotide to a discrete chromosomal location in intact nuclei are provided herein in the Detailed Description. The term "naturally-occurring@ as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polynucleotide sequence that is present in an organism 20 (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. A metabolically-active cell is a cell, comprising an intact nucleoid or nucleus, which, when provided nutrients and incubated in an appropriate medium carries out DNA synthesis and RNA for extended periods (e.g., at least 12-24 hours). Such metabolically-active cells are typically undifferentiated or 25 differentiated cells capable or incapable of further cell division (although non-dividing cells many undergo nuclear division and chromosomal replication), although stem cells and progenitor cells are also metabolically-active cells. As used herein, the term "disease allele@ refers to an allele of a gene which is capable of producing a recognizable disease. A disease allele may be dominant or recessive and may produce disease 30 directly or when present in combination with a specific genetic background or pre-existing pathological condition. A disease allele may be present in the gene pool or may be generated de novo in an individual by somatic mutation. For example and not limitation, disease to alleles include: activated oncogenes, a sickle cell anemia allele, a Tay-Sachs allele, a cystic fibrosis allele, a Lesch-Nyhan allele, a retinoblastoma-susceptibility allele, a Fabry's disease allele, and a Huntington's chorea allele. 19 WO 99/60108 PCT/US99/10731 As used herein, a disease allele encompasses both alleles associated with human diseases and alleles associated with recognized veterinary diseases. For example, the AF508 CFTR allele in a human disease allele which is associated with cystic fibrosis in North Americans. As used herein, the term "cell-uptake component@ refers to an agent which, when bound, either 5 directly or indirectly, to a targeting polynucleotide, enhances the intracellular uptake of the targeting polynucleotide into at least one cell type (e.g., hepatocytes). A cell-uptake component may include, but is not limited to, the following: specific cell surface receptors such as a galactose-terminal (asialo-) glycoprotein capable of being internalized into hepatocytes via a hepatocyte asialoglycoprotein receptor, a polycation (e.g., poly-L-lysine), and/or a protein-lipid complex formed with the targeting 10 polynucleotide. Various combinations of the above, as well as alternative cell-uptake components will be apparent to those of skill in the art and are provided in the published literature. DETAILED DESCRIPTION Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and 15 commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgenesis. Generally enzymatic reactions, oligonucleotide synthesis, oligonucleotide modification, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided 20 throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. Transgenic mice are derived according to Hogan, et al., "Manipulating the Mouse Embryo: A Laboratory Manual@, Cold Spring Harbor Laboratory (1988) which is incorporated herein by 25 reference. Embryonic stem cells are manipulated according to published procedures (Teratocarcinomas and embryonic stem cells: a practical approach, E.J. Robertson, ed., IRL Press, Washington, D.C., 1987; Zjilstra et al., Nature 342:435-438 (1989); and Schwartzberg et al., Science 246:799-803 (1989), each of which is incorporated herein by reference). 20 WO 99/60108 PCT/US99/10731 Zygotes are manipulated according to known procedures; for example see U.S. Patent No. 4,873,191, Brinster et al., PNAS 86:7007 (1989); Susulic et al., J. Biol. Chem. 49:29483 (1995), and Cavard et al., Nucleic Acids Res. 16:2099 (1988), hereby incorporated by reference. Oligonucleotides can be synthesized on an Applied Bio Systems oligonucleotide synthesizer according 5 to specifications provided by the manufacturer. Modified oligonucleotides and peptide nucleic acids are made as is generally known in the art. The present invention provides methods for targeting and altering, by homologous recombination, a pre-selected target nucleic acid sequence in a target cell, to make targeted sequence modifications. The methods comprise introducing into the target cells a recombinase and at least two single-stranded 0 targeting polynucleotides which are substantially complementary to each other. The targeting polynucleotides each comprise at least one homology clamp that substantially corresponds to or is substantially complementary to the preselected target nucleic acid sequence. The target cells are then screened to identify target cells containing the targeted sequence modification. Targeting Polynucleotides 5 Targeting polynucleotides may be produced by chemical synthesis of oligonucleotides, nick-translation of a double-stranded DNA template, polymerase chain-reaction amplification of a sequence (or ligase chain reaction amplification), purification of prokaryotic or target cloning vectors harboring a sequence of interest (e.g., a cloned cDNA or genomic clone, or portion thereof) such as plasmids, phagemids, YACs, cosmids, bacteriophage DNA, other viral DNA or replication intermediates, or purified restriction 20 fragments thereof, as well as other sources of single and double-stranded polynucleotides having a desired nucleotide sequence. Targeting polynucleotides are generally ssDNA or dsDNA, most preferably two complementary single-stranded DNAs. Targeting polynucleotides are generally at least about 2 to 100 nucleotides long, preferably at least about 5-to 100 nucleotides long, at least about 250 to 500 nucleotides long, more preferably at least 25 about 500 to 2000 nucleotides long, or longer; however, as the length of a targeting polynucleotide increases beyond about 20,000 to 50,000 to 400,000 nucleotides, the efficiency or transferring an intact targeting polynucleotide into the cell decreases. The length of homology may be selected at the discretion of the practitioner on the basis of the sequence composition and complexity of the predetermined endogenous target DNA sequence(s) and guidance provided in the art, which generally 30 indicates that 1.3 to 6.8 kilobase segments of homology are preferred (Hasty et al. (1991) Molec. Cell. Biol. 11: 5586; Shulman et al. (1990) Molec. Cell. Biol. 10: 4466, which are incorporated herein by reference). Targeting polynucleotides have at least one sequence that substantially corresponds to, or is substantially complementary to, a predetermined endogenous DNA sequence (i.e., a DNA 21 WO 99/60108 PCT/US99/10731 sequence of a polynucleotide located in a target cell, such as a chromosomal, mitochondrial, chloroplast, viral, episomal, or mycoplasmal polynucleotide). Such targeting polynucleotide sequences serve as templates for homologous pairing with the predetermined endogenous sequence(s), and are also referred to herein as homology clamps. In targeting polynucleotides, such 5 homology clamps are typically located at or near the 5' or 3' end, preferably homology clamps are internally or located at each end of the polynucleotide (Berinstein et al. (1992) Molec, Cell. Biol. 12: 360, which is incorporated herein by reference). Without wishing to be bound by any particular theory, it is believed that the addition of recombinases permits efficient gene targeting with targeting polynucleotides having short (i.e., about 50 to 1000 basepair long) segments of homology, as well as 10 with targeting polynucleotides having longer segments of homology. Therefore, it is preferred that targeting polynucleotides of the invention have homology clamps that are highly homologous to the predetermined target endogenous DNA sequence(s), most preferably isogenic. Typically, targeting polynucleotides of the invention have at least one homology clamp that is at least about 18 to 35 nucleotides long, and it is preferable that homology clamps are at least about 15 20 to 100 nucleotides long, and more preferably at least about 100-500 nucleotides long, although the degree of sequence homology between the homology clamp and the targeted sequence and the base composition of the targeted sequence will determine the optimal and minimal clamp lengths (e.g., G-C rich sequences are typically more thermodynamically stable and will generally require shorter clamp length). Therefore, both homology clamp length and the degree of sequence homology can only be 20 determined with reference to a particular predetermined sequence, but homology clamps generally must be at least about 12 nucleotides long and must also substantially correspond or be substantially complementary to a predetermined target sequence. Preferably, a homology clamp is at least about 12, and preferably at least about 50 nucleotides long and is identical to or complementary to a predetermined target sequence. Without wishing to be bound by a particular theory, it is believed that 25 the addition of recombinases to a targeting polynucleotide enhances the efficiency of homologous recombination between homologous, nonisogenic sequences (e.g., between an exon 2 sequence of a albumin gene of a Balb/c mouse and a homologous albumin gene exon 2 sequence of a C57/BL6 mouse), as well as between isogenic sequences. The formation of heteroduplex joints is not a stringent process; genetic evidence supports the view 30 that the classical phenomena of meiotic gene conversion and aberrant meiotic segregation result in part from the inclusion of mismatched base pairs in heteroduplex joints, and the subsequent correction of some of these mismatched base pairs before replication. Observations on recA protein have provided information on parameters that affect the discrimination of relatedness from perfect or near perfect homology and that affect the inclusion of mismatched base pairs in heteroduplex joints. The 35 ability of recA protein to drive strand exchange past all single base-pair mismatches and to form 22 WO 99/60108 PCT/US99/10731 extensively mismatched joints in superhelical DNA reflect its role in recombination and gene conversion. This error-prone process may also be related to its role in mutagenesis. RecA-mediated pairing reactions involving DNA of NX174 and G4, which are about 70 percent homologous, have yielded homologous recombinants (Cunningham et al. (1981) Cell 24: 213), although recA 5 preferentially forms homologous joints between highly homologous sequences, and is implicated as mediating a homology search process between an invading DNA strand and a recipient DNA strand, producing relatively stable heteroduplexes at regions of high homology. Accordingly, it is the fact that recombinases can drive the homologous recombination reaction between strands which are significantly, but not perfectly, homologous, which allows gene conversion and the modification of 10 target sequences. Thus, targeting polynucleotides may be used to introduce nucleotide substitutions, insertions and deletions into an endogeneous DNA sequence, and thus the corresponding amino acid substitutions, insertions and deletions in proteins expressed from the endogeneous DNA sequence. In a preferred embodiment, two substantially complementary targeting polynucleotides are used. In one embodiment, the targeting polynucleotides form a double stranded hybrid, which may be coated 15 with recombinase, although when the recombinase is recA, the loading conditions may be somewhat different from those used for single stranded nucleic acids. In a prefered embodiment, two substantially complementary single-stranded targeting polynucleotides are used. The two complementary single-stranded targeting polynucleotides are usually of equal length, although this is not required. However, as noted below, the stability of the four strand hybrids 20 of the invention is putatively related, in part, to the lack of significant unhybridized single-stranded nucleic acid, and thus significant unpaired sequences are not preferred. Furthermore, as noted above, the complementarity between the two targeting polynucleotides need not be perfect. The two complementary single-stranded targeting polynucleotides are simultaneously or contemporaneously introduced into a target cell harboring a predetermined endogenous target sequence, generally with at 25 lease one recombinase protein (e.g., recA). Under most circumstances, it is preferred that the targeting polynucleotides are incubated with recA or other recombinase prior to introduction into a target cell, so that the recombinase protein(s) may be "loaded" onto the targeting polynucleotide(s), to coat the nucleic acid, as is described below. Incubation conditions for such recombinase loading are described infra, and also in U.S.S.N. 07/755,462, filed 4 September 1991; U.S.S.N. 07/910,791, filed 9 30 July 1992; and U.S.S.N. 07/520,321, filed 7 May 1990, each of which is incorporated herein by reference. A targeting polynucleotide may contain a sequence that enhances the loading process of a recombinase, for example a recA loading sequence is the recombinogenic and recombinase nucleation sequence poly[d(A-C)] and its complement, poly[d(G-T)]. The duplex sequence oligo[d(A C), -d(G-T)n], where n is from 4 to 35, is a middle repetitive element in target DNA. 23 WO 99/60108 PCT/US99/10731 There appears to be a fundamental difference in the stability of RecA-protein-mediated D-loops formed between one single-stranded DNA (ssDNA) probe hybridized to negatively supercoiled DNA targets in comparison to relaxed or linear duplex DNA targets. Internally located dsDNA target sequences on relaxed linear DNA targets hybridized by ssDNA probes produce single D-loops, which are unstable 5 after removal of RecA protein (Adzuma, Genes Devel. 6:1679 (1992); Hsieh et al, PNAS USA 89:6492 (1992); Chiu et al., Biochemistry 32:13146 (1993)). This probe DNA instability of hybrids formed with linear duplex DNA targets is most probably due to the incoming ssDNA probe W-C base pairing with the complementary DNA strand of the duplex target and disrupting the base pairing in the other DNA strand. The required high free-energy of maintaining a disrupted DNA strand in an unpaired ssDNA 10 conformation in a protein-free single-D-loop apparently can only be compensated for either by the stored free energy inherent in negatively supercoiled DNA targets or by base pairing initiated at the distal ends of the joint DNA molecule, allowing the exchanged strands to freely intertwine. However, the addition of a second complementary ssDNA to the three-strand-containing single-D-loop stabilizes the deproteinized hybrid joint molecules by allowing W-C base pairing of the probe with the 15 displaced target DNA strand. The addition of a second RecA-coated complementary ssDNA (cssDNA) strand to the three-strand containing single D-loop stabilizes deproteinized hybrid joints located away from the free ends of the duplex target DNA (Sena & Zarling, Nature Genetics 3:365 (1993); Revet et al. J. Mol. Biol. 232:779 (1993); Jayasena and Johnston, J. Mol. Bio. 230:1015 (1993)). The resulting four-stranded structure, named a double D-loop by analogy with the three 20 stranded single D-loop hybrid has been shown to be stable in the absence of RecA protein. This stability likely occurs because the restoration of W-C basepairing in the parental duplex would require disruption of two W-C basepairs in the double-D-loop (one W-C pair in each heteroduplex D-loop). Since each base-pairing in the reverse transition (double-D-loop to duplex) is less favorable by the energy of one W-C basepair, the pair of cssDNA probes are thus kinetically trapped in duplex DNA 25 targets in stable hybrid structures. The stability of the double-D loop joint molecule within internally located probe:target hybrids is an intermediate stage prior to the progression of the homologous recombination reaction to the strand exchange phase. The double D-loop permits isolation of stable multistranded DNA recombination intermediates. In addition, when the targeting polynucleotides are used to generate insertions or deletions in an 30 endogeneous nucleic acid sequence, the use of two complementary single-stranded targeting polynucleotides allows the use of internal homology clamps as depicted in Figure 13. The use of internal homology clamps allows the formation of stable deproteinized cssDNA:probe target hybrids with homologous DNA sequences containing either relatively small or large insertions and deletions within a homologous DNA target. Without being bound by theory, it appears that these probe:target 35 hybrids, with heterologous inserts in the cssDNA probe, are stabilized by the re-annealing of cssDNA 24 WO 99/60108 PCT/US99/10731 probes to each other within the double-D-loop hybrid, forming a novel DNA structure with an internal homology clamp. Similarly stable double-D-loop hybrids formed at internal sites with heterologous inserts in the linear DNA targets (with respect to the cssDNA probe) are equally stable. Because cssDNA probes are kinetically trapped within the duplex target, the multi-stranded DNA intermediates 5 of homologous DNA pairing are stabilized and strand exchange is facilitated. In a preferred embodiment, the length of the internal homology clamp (i.e. the length of the insertion or deletion) is from about 1 to 50% of the total length of the targeting polynucleotide, with from about 1 to about 20% being preferred and from about 1 to about 10% being especially preferred, although in some cases the length of the deletion or insertion may be significantly larger. As for the targeting 10 homology clamps, the complementarity within the internal homology clamp need not be perfect. The invention may also be practiced with individual targeting polynucleotides which do not comprise part of a complementary pair. In each case, a targeting polynucleotide is introduced into a target cell simultaneously or contemporaneously with a recombinase protein, typically in the form of a recombinase coated targeting polynucleotide as outlined herein (i.e., a polynucleotide pre-incubated 15 with recombinase wherein the recombinase is noncovalently bound to the polynucleotide; generally referred to in the art as a nucleoprotein filament). A targeting polynucleotide used in a method of the invention typically is a single-stranded nucleic acid, usually a DNA strand, or derived by denaturation of a duplex DNA, which is complementary to one (or both) strand(s) of the target duplex nucleic acid. Thus, one of the complementary single stranded 20 targeting polynucleotides is complementary to one strand of the endogeneous target sequence (i.e. Watson) and the other complementary single stranded targeting polynucleotide is complementary to the other strand of the endogeneous target sequence (i.e. Crick). The homology clamp sequence preferably contains at least 90-95% sequence homology with the target sequence, to insure sequence-specific targeting of the targeting polynucleotide to the endogenous DNA target. Each 25 single-stranded targeting polynucleotide is typically about 50-600 bases long, although a shorter or longer polynucleotide may also be employed. Alternatively, targeting polynucleotides may be prepared in single-stranded form by oligonucleotide synthesis methods, which may first require, especially with larger targeting polynucleotides, formation of subfragments of the targeting polynucleotide, typically followed by splicing of the subfragments together, typically by enzymatic 30 ligation. Recombinase Proteins 25 WO 99/60108 PCT/US99/10731 Recombinases are proteins that, when included with an exogenous targeting polynucleotide, provide a measurable increase in the recombination frequency and/or localization frequency between the targeting polynucleotide and an endogenous predetermined DNA sequence. Thus, in a preferred embodiment, increases in recombination frequency from the normal range of 10-8 to 104, to 104 to 101, 5 preferably 104 to 101, and most preferably 10-2 to 101, may be acheived. In the present invention, recombinase refers to a family of RecA-like recombination proteins all having essentially all or most of the same functions, particularly: (i) the recombinase protein's ability to properly bind to and position targeting polynucleotides on their homologous targets and (ii) the ability of recombinase protein/targeting polynucleotide complexes to efficiently find and bind to 10 complementary endogenous sequences. The best characterized recA protein is from E. coli, in addition to the wild-type protein a number of mutant recA-like proteins have been identified (e.g., recA803; see Madiraju et al., PNAS USA 85(18):6592 (1988); Madiraju et al, Biochem. 31:10529 (1992); Lavery et al., J. Biol. Chem. 267:20648 (1992)). Further, many organisms have recA-like recombinases with strand-transfer activities (e.g., Fugisawa et al., (1985) Nucl. Acids Res. 13: 7473; 15 Hsieh et al., (1986) Cell 44: 885; Hsieh et al., (1989) J. Biol. Chem. 264: 5089; Fishel et al., (1988) Proc. Nati. Acad. Sci. (USA) 85: 3683; Cassuto et al., (1987) Mol. Gen. Genet. 208: 10; Ganea et al., (1987) Mol. Cell Biol. 7: 3124; Moore et al., (1990) . Biol. Chem. 19: 11108; Keene et al., (1984) Nuc. Acids Res. 12: 3057; Kimeic, (1984) Cold Spring Harbor Svmp. 48: 675; Kmeic, (1986) Cel: 44 545; Kolodner et al., (1987) Proc. Nati. Acad. Sci. USA 84: 5560; Sugino et al., (1985) Proc. Natl. Acad. Sci. 20 USA 85: 3683; Halbrook et al., (1989) J. Biol. Chem. 264: 21403; Eisen et al., (1988) Proc. NatI. Acad. Sci. USA 85: 7481; McCarthy et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5854; Lowenhaupt et al., (1989) J. Biol. Chem. 264: 20568, which are incorporated herein by reference. Examples of such recombinase proteins include, for example but not limitation: recA, recA803, uvsX, and other recA mutants and recA-like recombinases (Roca, A. i. (1990) Crit. Rev. Biochem. Molec. Biol. 25: 415), 25 seol (Kolodner et al. (1987) Proc. NatI. Acad. Sci. (U.S.A.) 84:5560; Tishkoff et al. Molec. Cell. Biol. 11:2593), RuvC (Dunderdale et al. (1991) Nature 354: 506), DST2, KEM1, XRN1 (Dykstra et al. (1991) Molec. Cell. Biol. 11:2583), STP"/DST1 (Clark et al. (1991) Molec. Cell. Biol. 11:2576), HPP-1 (Moore et al. (1991) Proc. NatI. Acad. Sci. (U.S.A.) 88:9067), other target recombinases (Bishop et al. (1992) Cell 69: 439; Shinohara et al. (1992) Cel 69: 457); incorporated herein by reference. RecA 30 may be purified from E coli strains, such as E. coli strains JC12772 and JC15369 (available from A.J. Clark and M. Madiraju, University of California-Berkeley, or purchased commercially). These strains contain the recA coding sequences on a "runaway" replicating plasmid vector present at a high copy numbers per cell. The recA803 protein is a high-activity mutant of wild-type recA. The art teaches several examples of recombinase proteins, for example, from Drosophila, yeast, plant, human, and 35 non-human mammalian cells, including proteins with biological properties similar to recA (i.e., recA-like recombinases), such as Rad5l from mammals and yeast, and Pk-rec (see Rashid et al., Nucleic Acid 26 WO 99/60108 PCTIUS99/10731 Res. 25(4):719 (1997), hereby incorporated by reference). In addition, the recombinase may actually be a complex of proteins, i.e. a Arecombinosome@. In addition, included within the definition of a recombinase are portions or fragments of recombinases which retain recombinase biological activity, as well as variants or mutants of wild-type recombinases which retain biological activity, such as the E. 5 coli recA803 mutant with enhanced recombinase activity. In a preferred embodiment, recA or rad5l is used. For example, recA protein is typically obtained from bacterial strains that overproduce the protein: wild-type E. coli recA protein and mutant recA803 protein may be purified from such strains. Alternatively, recA protein can also be purchased from, for example, Pharmacia (Piscataway, NJ). 10 RecA proteins, and its homologs, form a nucleoprotein filament when it coats a single-stranded DNA. In this nucleoprotein filament, one monomer of recA protein is bound to about 3 nucleotides. This property of recA to coat single-stranded DNA is essentially sequence independent, although particular sequences favor initial loading of recA onto a polynucleotide (e.g., nucleation sequences). The nucleoprotein filament(s) can be formed on essentially any DNA molecule and can be formed in cells 15 (e.g., mammalian cells), forming complexes with both single-stranded and double-stranded DNA, although the loading conditions for dsDNA are somewhat different than for ssDNA. Recombinase Coating of Targeting Polynucleotides The conditions used to coat targeting polynucleotides with recombinases such as recA protein and ATP(S have been described in commonly assigned U.S.S.N. 07/910,791, filed 9 July 1992; U.S.S.N. 20 07/755,462, filed 4 September 1991; and U.S.S.N. 07/520,321, filed 7 May 1990, each incorporated herein by reference. The procedures below are directed to the use of E. coli recA, although as will be appreciated by those in the art, other recombinases may be used as well. Targeting polynucleotides can be coated using GTP(S, mixes of ATP(S with rATP, rGTP and/or dATP, or dATP or rATP alone in the presence of an rATP generating system (Boehringer Mannheim). Various mixtures of GTP(S, 25 ATP(S, ATP, ADP, dATP and/or rATP or other nucleosides may be used, particularly preferred are mixes of ATP(S and ATP or ATP(S and ADP. RecA protein coating of targeting polynucleotides is typically carried out as described in U.S.S.N. 07/910,791, filed 9 July 1992 and U.S.S.N. 07/755,462, filed 4 September 1991, which are incorporated herein by reference. Briefly, the targeting polynucleotide, whether double-stranded or 30 single-stranded, is denatured by heating in an aqueous solution at 95-1 00*C for five minutes, then placed in an ice bath for 20 seconds to about one minute followed by centrifugation at 0*C for approximately 20 sec, before use. When denatured targeting polynucleotides are not placed in a 27 WO 99/60108 PCT/US99/10731 freezer at -20 0 C they are usually immediately added to standard recA coating reaction buffer containing ATP(S, at room temperature, and to this is added the recA protein. Alternatively, recA protein may be included with the buffer components and ATP(S before the polynucleotides are added. RecA coating of targeting polynucleotide(s) is initiated by incubating polynucleotide-recA mixtures at 5 370C for 10-15 min. RecA protein concentration tested during reaction with polynucleotide varies depending upon polynucleotide size and the amount of added polynucleotide, and the ratio of recA molecule:nucleotide preferably ranges between about 3:1 and 1:3. When single-stranded polynucleotides are recA coated independently of their homologous polynucleotide strands, the mM and pM concentrations of ATP(S and recA, respectively, can be reduced to one-half those used with 10 double-stranded targeting polynucleotides (i.e., recA and ATP(S concentration ratios are usually kept constant at a specific concentration of individual polynucleotide strand, depending on whether a single or double-stranded polynucleotide is used). RecA protein coating of targeting polynucleotides is normally carried out in a standard 1X RecA coating reaction buffer. 1OX RecA reaction buffer (i.e., 10x AC buffer) consists of: 100 mM Tris 15 acetate (pH 7.5 at 370C), 20 mM magnesium acetate, 500 mM sodium acetate, 10 mM DTT, and 50% glycerol). All of the targeting polynucleotides, whether double-stranded or single-stranded, typically are denatured before use by heating to 95-100*C for five minutes, placed on ice for one minute, and subjected to centrifugation (10,000 rpm) at 0*C for approximately 20 seconds (e.g., in a Tomy centrifuge). Denatured targeting polynucleotides usually are added immediately to room temperature 20 RecA coating reaction buffer mixed with ATP(S and diluted with buffer or double-distilled H 2 0 as necessary. A reaction mixture typically contains the following components: (i) 0.2-4.8 mM ATP(S; and (ii) between 1-100 ng/pl of targeting polynucleotide. To this mixture is added about 1-20 pl of recA protein per 10 100 pl of reaction mixture, usually at about 2-10 mg/ml (purchased from Pharmacia or purified), and is 25 rapidly added and mixed. The final reaction volume-for RecA coating of targeting polynucleotide is usually in the range of about 10-500 pl. RecA coating of targeting polynucleotide is usually initiated by incubating targeting polynucleotide-RecA mixtures at 370C for about 10-15 min. RecA protein concentrations in coating reactions varies depending upon targeting polynucleotide size and the amount of added targeting polynucleotide: recA protein concentrations are typically in the 30 range of 5 to 50 pM. When single-stranded targeting polynucleotides are coated with recA, independently of their complementary strands, the concentrations of ATP(S and recA protein may optionally be reduced to about one-half of the concentrations used with double-stranded targeting 28 WO 99/60108 PCT/US99/10731 polynucleotides of the same length: that is, the recA protein and ATP(S concentration ratios are generally kept constant for a given concentration of individual polynucleotide strands. The coating of targeting polynucleotides with recA protein can be evaluated in a number of ways. First, protein binding to DNA can be examined using band-shift gel assays (McEntee et al., (1981)1 5 Biol. Chem. 256: 8835). Labeled polynucleotides can be coated with recA protein in the presence of ATP(S and the products of the coating reactions may be separated by agarose gel electrophoresis. Following incubation of recA protein with denatured duplex DNAs the recA protein effectively coats single-stranded targeting polynucleotides derived from denaturing a duplex DNA. As the ratio of recA protein monomers to nucleotides in the targeting polynucleotide increases from 0, 1:27, 1:2.7 to 3.7:1 10 for 121-mer and 0, 1:22, 1:2.2 to 4.5:1 for 159-mer, targeting polynucleotide's electrophoretic mobility decreases, i.e., is retarded, due to recA-binding to the targeting polynucleotide. Retardation of the coated polynucleotide's mobility reflects the saturation of targeting polynucleotide with recA protein. An excess of recA monomers to DNA nucleotides is required for efficient recA coating of short targeting polynucleotides (Leahy et al., (1986) J. Biol. Chem. 261: 954). 15 A second method for evaluating protein binding to DNA is in the use of nitrocellulose filter binding assays (Leahy et al., (1986) J. Biol. Chem. 261:6954; Woodbury, et al., (1983) Biochemistry 22(20):4730-4737. The nitrocellulose filter binding method is particularly useful in determining the dissociation-rates for protein:DNA complexes using labeled DNA. In the filter binding assay, DNA:protein complexes are retained on a filter while free DNA passes through the filter. This assay 20 method is more quantitative for dissociation-rate determinations because the separation of DNA:protein complexes from free targeting polynucleotide is very rapid. Alternatively, recombinase protein(s) (prokaryotic, eukaryotic or endogeneous to the target cell) may be exogenously induced or administered to a target cell simultaneously or contemporaneously (i.e., within about a few hours) with the targeting polynucleotide(s). Such administration is typically done by 25 micro-injection, although electroporation, lipofection, and other transfection methods known in the art may also be used. Alternatively, recombinase-proteins may be produced in vivo. For example, they may be produced from a homologous or heterologous expression cassette in a transfected cell or transgenic cell, such as a transgenic totipotent cell (e.g. a fertilized zygote) or an embryonal stem cell (e.g., a murine ES cell such as AB-1) used to generate a transgenic non-human animal line or a 30 somatic cell or a pluripotent hematopoietic stem cell for reconstituting all or part of a particular stem cell population (e.g. hematopoietic) of an individual. Conveniently, a heterologous expression cassette includes a modulatable promoter, such as an ecdysone-inducible promoter-enhancer combination, an estrogen-induced promoter-enhancer combination, a CMV promoter-enhancer, an insulin gene promoter, or other cell-type specific, developmental stage-specific, hormone-inducible, or other 29 WO 99/60108 PCT/US99/10731 modulatable promoter construct so that expression of at least one species of recombinase protein from the cassette can by modulated for transiently producing recombinase(s) in vivo simultaneous or contemporaneous with introduction of a targeting polynucleotide into the cell. When a hormone-inducible promoter-enhancer combination is used, the cell must have the required hormone 5 receptor present, either naturally or as a consequence of expression a co-transfected expression vector encoding such receptor. Alternatively, the recombinase may be endogeneous and produced in high levels. In this embodiment, preferably in eukaryotic target cells such as tumor cells, the target cells produce an elevated level of recombinase. In other embodiments the level of recombinase may be induced by DNA damaging agents, such as mitomycin C, UV or (-irradiation. Alternatively, 10 recombinase levels may also be elevated by transfection of a virus or plasmid encoding the recombinase gene into the cell. Cell-Uptake Components A targeting polynucleotide of the invention may optionally be conjugated, typically by covalently or preferably noncovalent binding, to a cell-uptake component. Various methods have been described in 15 the art for targeting DNA to specific cell types. A targeting polynucleotide of the invention can be conjugated to essentially any of several cell-uptake components known in the art. For targeting to hepatocytes, a targeting polynucleotide can be conjugated to an asialoorosomucoid (ASOR)-poly-L lysine conjugate by methods described in the art and incorporated herein by reference (Wu GY and Wu CH (1987) J. Biol. Chem. 262:4429; Wu GY and Wu CH (1988) Biochemistry 27:887; Wu GY and 20 Wu CH (1988) J. Biol. Chem. 263: 14621; Wu GY and Wu CH (1992) J. Biol. Chem. 267 12436; Wu et al. (1991) J. Biol. Chem. 266: 14338; and Wilson et al. (1992) J. Biol. Chem. 267: 963, W092/06180; W092/05250; and W091/17761, which are incorporated herein by reference). Alternatively, a cell-uptake component may be formed by incubating the targeting polynucleotide with at least one lipid species and at least one protein species to form protein-lipid-polynucleotide 25 complexes consisting essentially of the targeting polynucleotide and the lipid-protein cell-uptake component. Lipid vesicles made according to Feigner (W091/17424, incorporated herein by reference) and/or cationic lipidization (WO91/16024, incorporated herein by reference) or other forms for polynucleotide administration (EP 465,529, incorporated herein by reference) may also be employed as cell-uptake components. Nucleases may also be used. 30 In addition to cell-uptake components, targeting components such as nuclear localization signals may be used, as is known in the art. Homologous Pairing of Targetinq Polynucleotides Having Chemical Substituents 30 WO 99/60108 PCTIUS99/10731 In addition to recombinase and cellular uptake components, the targeting polynucleotides may include chemical substituents. Exogenous targeting polynucleotides that have been modified with appended chemical substituents may be introduced along with recombinase (e.g., recA) into a metabolically active target cell to homologously pair with a predetermined endogenous DNA target sequence in the 5 cell. In a preferred embodiment, the exogenous targeting polynucleotides are derivatized, and additional chemical substituents are attached, either during or after polynucleotide synthesis, respectively, and are thus localized to a specific endogenous target sequence where they produce an alteration or chemical modification to a local DNA sequence. Preferred attached chemical substituents include, but are not limited to: cross-linking agents (see Podyminogin et al., Biochem. 34:13098 10 (1995) and 35:7267 (1996), both of which are hereby incorporated by reference), nucleic acid cleavage agents, metal chelates (e.g., iron/EDTA chelate for iron catalyzed cleavage), topoisomerases, endonucleases, exonucleases, ligases, phosphodiesterases, photodynamic porphyrins, chemotherapeutic drugs (e.g., adriamycin, doxirubicin), intercalating agents, labels, base modification agents, agents which normally bind to nucleic acids such as labels, etc. (see for example 15 Afonina et al., PNAS USA 93:3199 (1996), incorporated herein by reference) immunoglobulin chains, and oligonucleotides. Iron/EDTA chelates are particularly preferred chemical substituents where local cleavage of a DNA sequence is desired (Hertzberg et al. (1982) J. Am. Chem. Soc. 104: 313; Hertzberg and Dervan (1984) Biochemistry 23: 3934; Taylor et al. (1984) Tetrahedron 40: 457; Dervan, PB ( 1986) Science 232: 464, which are incorporated herein by reference). Further preferred 20 are groups that prevent hybridization of the complementary single stranded nucleic acids to each other but not to unmodified nucleic acids; see for example Kutryavin et al., Biochem. 35:11170 (1996) and Woo et al., Nucleic Acid. Res. 24(13):2470 (1996), both of which are incorporated by reference. 2'-0 methyl groups are also preferred; see Cole-Strauss et al., Science 273:1386 (1996); Yoon et al., PNAS 93:2071 (1996)). Additional preferred chemical substitutents include labeling moieties, 25 including fluorescent labels. Preferred attachment chemistries include: direct linkage, e.g., via an appended reactive amino group (Corey and Schultz (1988) Science 238:1401, which is incorporated herein by reference) and other direct linkage chemistries, although streptavidin/biotin and digoxigenin/antidigoxigenin antibody linkage methods may also be used. Methods for linking chemical substituents are provided in U.S. Patents 5,135,720, 5,093,245, and 5,055,556, which are 30 incorporated herein by reference. Other linkage chemistries may be used at the discretion of the practitioner. Typically, a targeting polynucleotide of the invention is coated with at least one recombinase and is conjugated to a cell-uptake component, and the resulting cell targeting complex is contacted with a target cell under uptake conditions (e.g., physiological conditions) so that the targeting polynucleotide 35 and the recombinase(s) are internalized in the target cell. A targeting polynucleotide may be contacted simultaneously or sequentially with a cell-uptake component and also with a recombinase; 31 WO 99/60108 PCT/US99/10731 preferably the targeting polynucleotide is contacted first with a recombinase, or with a mixture comprising both a cell-uptake component and a recombinase under conditions whereby, on average, at least about one molecule of recombinase is noncovalently attached per targeting polynucleotide molecule and at least about one cell-uptake component also is noncovalently attached. Most 5 preferably, coating of both recombinase and cell-uptake component saturates essentially all of the available binding sites on the targeting polynucleotide. A targeting polynucleotide may be preferentially coated with a cell-uptake component so that the resultant targeting complex comprises, on a molar basis, more cell-uptake component than recombinase(s). Alternatively, a targeting polynucleotide may be preferentially coated with recombinase(s) so that the resultant targeting 10 complex comprises, on a molar basis, more recombinase(s) than cell-uptake component. Cell-uptake components are included with recombinase-coated targeting polynucleotides of the invention to enhance the uptake of the recombinase-coated targeting polynucleotide(s) into cells, particularly for in yvv gene targeting applications, such as gene therapy to treat genetic diseases, including neoplasia, and targeted homologous recombination to treat viral infections wherein a viral 15 sequence (e.g., an integrated hepatitis B virus (HBV) genome or genome fragment) may be targeted by homologous sequence targeting and inactivated. Alternatively, a targeting polynucleotide may be coated with the cell-uptake component and targeted to cells with a contemporaneous or simultaneous administration of a recombinase (e.g., liposomes or immunoliposomes containing a recombinase, a viral-based vector encoding and expressing a recombinase). 20 Once the recombinase-targeting polynucleotide compositions are formulated, they are introduced or administered into target cells. The administration is typically done as is known for the administration of nucleic acids into cells, and, as those skilled in the art will appreciate, the methods may depend on the choice of the target cell. Suitable methods include, but are not limited to, microinjection, electroporation, lipofection, etc. By Atarget cells@ herein is meant prokaryotic or eukaryotic cells. 25 Suitable prokaryotic cells include, but are not limited to, bacteria such as E. coli, Bacillus species, and the extremophile bacteria such as thermophiles, etc. Preferably, the procaryotic target cells are recombination competent. Suitable eukaryotic cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Aspergillus, Trichoderma, and Neurospora; plant cells including those of corn, sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa, sunflower, 30 etc.; and animal cells, including fish, birds and mammals. Suitable fish cells include, but are not limited to, those from species of salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish. Suitable bird cells include, but are not limited to, those of chickens, ducks, quail, pheasants and turkeys, and other jungle fowl or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cattle, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters, 35 gerbils, and guinea pigs, minks, goats, pigs, primates, marsupials, marine mammals including dolphins 32 WO 99/60108 PCT/US99/10731 and whales, as well as cell lines, such as human cell lines of any tissue or stem cell type, and stem cells, including pluripotent and non-pluripotent, and non-human zygotes. In a preferred embodiment, procaryotic cells are used. In this embodiment, a pre-selected target DNA sequence is chosen for alteration. Preferably, the pre-selected target DNA sequence is contained 5 within an extrachromosomal sequence. By Aextrachromosomal sequence@ herein is meant a sequence separate from the chromosomal or genomic sequences. Preferred extrachromosomal sequences include plasmids (particularly procaryotic plasmids such as bacterial plasmids), P1 vectors, viral genomes, yeast, bacterial and mammalian artificial chromosomes (YAC, BAC and MAC, respectively), and other autonomously self-replicating sequences, although this is not required. As 10 described herein, a recombinase and at least two single stranded targeting polynucleotides which are substantially complementary to each other, each of which contain a homology clamp to the target sequence contained on the extrachromosomal sequence, are added to the extrachromosomal sequence, preferably in vitro. The two single stranded targeting polynucleotides are preferably coated with recombinase, and at least one of the targeting polynucleotides contain at least one nucleotide 15 substitution, insertion or deletion. The targeting polynucleotides then bind to the target sequence in the extrachromosomal sequence to effect homologous recombination and form an altered extrachromosomal sequence which contains the substitution, insertion or deletion. The altered extrachromosomal sequence is then introduced into the procaryotic cell using techniques known in the art. Preferably, the recombinase is removed prior to introduction into the target cell, using techniques 20 known in the art. For example, the reaction may be treated with proteases such as proteinase K, detergents such as SDS, and phenol extraction (including phenol:chloroform:isoamyl alcohol extraction). These methods may also be used for eukaryotic cells. Alternatively, the pre-selected target DNA sequence is a chromosomal sequence. In this embodiment, the recombinase with the targeting polynucleotides are introduced into the target cell, preferably 25 eukaryotic target cells. In this embodiment, it may be desirable to bind (generally non-covalently) a nuclear localization signal to the targeting polynucleotides to facilitate localization of the complexes in the nucleus. See for example Kido et al., Exper. Cell Res. 198:107-114 (1992), hereby expressly incorporated by reference. The targeting polynucleotides and the recombinase function to effect homologous recombination, resulting in altered chromosomal or genomic sequences. 30 In a preferred embodiment, eukaryotic cells are used. For making transgenic non-human animals (which include homologously targeted non-human animals) embryonal stem cells (ES cells) and fertilized zygotes are preferred. In a preferred embodiment, embryonal stem cells are used. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62: 1073-1085 (1990)) essentially as described (Robertson, E.J. (1987) in 33 WO 99/60108 PCT/US99/10731 Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson, ed. (oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al. (1987) Nature 326: 292-295), the D3 line (Doetschman et al. (1985) J. Embryol. Exp. Morph. 8Z: 21-45), and the CCE line (Robertson et al. (1986) Nature 323: 5 445-448). The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i.e., their ability, once injected into a host blastocyst, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The pluripotence of any given ES cell line can vary with time in culture and the care with which it has been handled. The only definitive assay for pluripotence is to determine whether the specific 10 population of ES cells to be used for targeting can give rise to chimeras capable of germline transmission of the ES genome. For this reason, prior to gene targeting, a portion of the parental population of AB-1 cells is injected into C57B1/6J blastocysts to ascertain whether the cells are capable of generating chimeric mice with extensive ES cell contribution and whether the majority of these chimeras can transmit the ES genome to progeny. 15 In a preferred embodiment, non-human zygotes are used, for example to make transgenic animals, using techniques known in the art (see U.S. Patent No. 4,873,191). Preferred zygotes include, but are not limited to, animal zygotes, including fish, avian and mammalian zygotes. Suitable fish zygotes include, but are not limited to, those from species of salmon, trout, tuna, carp, flounder, halibut, swordfish, cod, tulapia and zebrafish. Suitable bird zygotes include, but are not limited to, those of 20 chickens, ducks, quail, pheasant, turkeys, and other jungle fowl and game birds. Suitable mammalian zygotes include, but are not limited to, cells from horses, cattle, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, and marine mammals including dolphins and whales. See Hogan et al., Manipulating the Mouse Embryo (A Laboratory Manual), 2nd Ed. Cold Spring Harbor Press, 1994, incorporated by reference. 25 Once made and administered to a target host cell, the compositions of the invention find use in a number of applications, including the creation of transgenic plants and animals. Such transgenic animals can be any of the animals, fish and birds outlined above as suitable for zygotes. Preferably the transgenic animals are mammals, including, but not limited to, farm animals such as cattle, buffalo, goats, including BELE@ goats, sheep, and pigs or other transgenic animals such as mice, rabbits, 30 monkeys, etc. In a preferred embodiment, the animals or mammals are non-human. In general, transgenic animals are made with any number of changes. Exogeneous sequences, or extra copies of endogeneous sequences, including structural genes and regulatory sequences, may be added to the animal, as outlined below. Endogeneous sequences (again, either genes or 34 WO 99/60108 PCT/US99/10731 regulatory sequences) may be disrupted, i.e. via insertion, deletion or substitution, to prevent expression of endogeneous proteins. Alternatively, endogeneous sequences may be modified to alter their biological function, for example via mutation of the endogeneous sequence by insertion, deletion or substitution. 5 Accordingly, tThe methods of the present invention are useful to add exogenous DNA sequences, such as exogenous genes or regulatory sequences, extra copies of endogenous genes or regulatory sequences, or exogeneous genes or regulatory sequences, to a transgenic plant or animal. This may be done for a number of reasons: for example, adding one or more copies of a wild-type gene can increase the production of a desirable gene product; adding or deleting one or more copies of a 10 therapeutic gene can alleviate a disease state, or to create an animal model of disease. Adding one or more copies of a modified wild type gene may be done for the same reasons. Adding therapeutic genes or proteins may yield superior transgenic animals, for example for the production of therapeutic or nutriceutical proteins. Adding human genes to non-human mammals may facilitate production of human proteins and adding regulatory sequences derived from human or non-human mammals may 15 be useful to increase or decrease the expression of endogenous or exogenous genes. Such inserted genes may be under the control of endogenous or exogenous regulatory sequences, as described herein. The methods of the invention are also useful to modify endogeneous gene sequences, as outlined below. Suitable endogenous gene targets include, but are not limited to, genes which encode 20 peptides or proteins including enzymes, structural or soluble proteins, as well as endogeneous regulatory sequences including, but not limited to, promoters, transcriptional or translational sequences, repetitive sequencs including oligo[d(A-C)n -d(G-T)], oligo[d(A-T)]n, oligo[d(C-T)]n, etc. Examples of such endogenous gene targets include, but are not limited to, genes which encode lactoglobulins including both a-lactoglobulin and $-lactoglobulin; casein, including both a-casein, 25 P-casein and K-casein; albumins, including serum albumin, particularly human and bovine; immunoglobulins, including IgE, IgM, IgG and igD and monoclonal antibodies; globin; integrin; hormones; growth factors, particularly bovine and human growth factors, including transforming growth factor, epidermal growth factor, nerve growth factors, etc.; collagen; interleukins, including IL-1 to IL 17; a major histocompatibility antigen (MHC); G-protein coupled receptors (GPCR); nuclear receptors; 30 ion channels; multidrug resistance genes; amyloid proteins; enzymes, including esterases, proteases (including tissue plasminogen activator (tPA)), lipases, carbohydrases, etc.; APRT, HPRT; leptin; tumor suppressor genes; provirus; prions; OTC; CFTR; sugar transferases such as alpha-galactosyl transferase (gaIT) or fucosyl transferase; a milk or urine protein gene including the caseins, lactoferrin and whey proteins; oncogenes; cytokines, particularly human; transcription factors; and other 35 WO 99/60108 PCTIUS99/10731 pharmaceuticals. Any or all of these may also be suitable exogeneous genes to add to a genome using the methods outlined herein. Endogeneous genes (or regulatory sequences, as outlined herein) may be modified in several ways, including disruptions and alterations. 5 The endogenous target gene may be disrupted in a variety of ways. The term Adisrupt@ as used herein comprises a change in the coding or non-coding sequence of an endogenous nucleic acid that alters the transcription or translation of an endogenous gene. In a preferred embodiment, a disrupted gene will no longer produce a functional gene product. Generally, disruption may occur by either the insertion, deletion or frame shifting of nucleotides. 10 The term Ainsertion sequence@ as used herein means one or more nucleotides which are inserted into an endogenous gene to disrupt it. In general, insertion sequences can be as short as 1 nucleotide or as long as a gene, as outlined below. For non-gene insertion sequences, the sequences are at least 1 nucleotide, with from about 1 to about 50 nucleotides being preferred, and from about 10 to 25 nucleotides being particularly preferred. An insertion sequence may comprise a polylinker sequence, 15 with from about 1 to about 50 nucleotides being preferred, and from about 10 to 25 nucleotides being particularly preferred. In a preferred embodiment, an insertion sequence comprises a gene which not only disrupts the endogenous gene, thus preventing its expression, but also can result in the expression of a new gene product. Thus, in a preferred embodiment, the disruption of an endogenous gene by an insertion 20 sequence gene is done in such a manner to allow the transcription and translation of the insertion gene. An insertion sequence that encodes a gene may range from about 50 bp to 5000 bp of cDNA or about 5000 bp to 50000 bp of genomic DNA. As will be appreciated by those in the art, this can be done in a variety of ways. In a preferred embodiment, the insertion gene is targeted to the endogenous gene in such a manner as to utilize endogenous regulatory sequences, including 25 promoters, enhancers or a regulatory sequence. In an alternate embodiment, the insertion sequence gene includes its own regulatory sequences, such as a promoter, enhancer or other regulatory sequence etc. Particularly preferred insertion sequence genes include, but are not limited to, genes which encode therapeutic and nutriceutical proteins, and reporter genes. Suitable insertion sequence genes which 30 may be inserted into endogenous genes include, but are not limited to, nucleic acids which encode those genes listed as suitable endogeneous genes for alterations, above, particularly mammalian enzymes, mammalian antibodies, mammalian proteins including serum albumin as well as mammalian 36 WO 99/60108 PCTIUS99/10731 therapeutic genes. In a preferred embodiment, the inserted mammalian gene is a human gene. Suitable reporter genes are those genes which encode detectable proteins, such as the genes encoding luciferase, P-galactosidase (both of which require the addition of reporter substrates), and the fluorescent proteins, including green fluorescent protein (GFP), blue fluorescent protein (BFP), 5 yellow fluorescent protein (YFP), and red fluorescent protein (RFP). Thus, in a preferred embodiment, the targeted sequence modification creates a sequence that has a biological activity or encodes a polypeptide having a biological activity. In a preferred embodiment, the polypeptide is an enzyme with enzymatic activity. In another preferred embodiment, the polypeptide is an antibody. In a third preferred embodiment, the polypeptide is a structural protein. 10 In addition, the insertion sequence genes may be modified or variant genes, i.e. they contain a mutation from the wild-type sequence. Thus, for example, modified genes including, but not limited to, improved therapeutic genes, modified "-lactalbumin genes that do not encode any phenylalanine residues, or human enzyme or human antibody genes that do not encode any phenylalanine residues. The term Adeletion@ as used herein comprises removal of a portion of the nucleic acid sequence of 15 an endogenous gene. Deletions range from about 1 to about 100 nucleotides, with from about 1 to 50 nucleotides being preferred and from about 1 to about 25 nucleotides being particularly preferred, although in some cases deletions may be much larger, and may effectively comprise the removal of the entire endogenous gene and/or its regulatory sequences. Deletions may occur in combination with substitutions or modifications to arrive at a final modified endogenous gene. 20 In a preferred embodiment, endogenous genes may be disrupted simultaneously by an insertion and a deletion. For example, some or all of an endogenous gene, with or without its regulatory sequences, may be removed and replaced with an insertion sequence gene. Thus, for example, all but the regulatory sequences of an endogenous gene may be removed, and replaced with an insertion sequence gene, which is now under the control of the endogenous gene's regulatory elements. 25 The term Aregulatory element@ is used herein to describe a non-coding sequence which affects the transcription or translation of a gene including, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, enhancer or activator sequences, or dimerizing sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequence. 37 WO 99/60108 PCT/US99/10731 Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In addition to disrupting endogeneous genes, the endogeneous genes may be altered by substitutions, 5 insertions or deletions of nucleotides that do not completely eliminate the biological function of the sequence, but rather alter it. That is, targeted gene modifications may be made to alter gene function. For example, defective genes may be fixed, or the activity of a gene may be modulated, either increasing or decreasing the activity of the sequence (either the nucleic acid sequence, for example in the case of regulatory nucleic acid, or of the gene product, i.e. the amino acid sequence of the protein 10 may be altered). The methods of the present invention are useful to provide methods for fully or partially modifying endogenous regulatory sequences. Suitable targets for such fully or partially modified regulatory sequences include, but are not limited to, regulatory sequences that regulate any of the suitable endogeneous genes listed above, with preferred embodiments altering the endogeneous regulatory 15 sequences that control the genes which encode "-lactoglobulin, $-lactoglobulin, casein, a-casein, p casein, K-casein, serum albumin, globin, IgG, integrin, lactoferrin, a retroviral provirus, a prion, alpha-galactosyl transferase (gaIT), a sugar transferase or a milk or urine production gene. Examples of such fully or partially modified endogenous regulatory sequences include, but are not limited to, a modified regulatory element for an endogenous gene, a modified transcriptional regulation cassette or 20 start site for an endogenous gene, a modified promoter, transcription initiation site, or enhancer sequences. When the modification of the endogeneous gene is to alter a structural gene, generally amino acid changes will be made as is known in the art. Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino 25 acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances or for certain purposes. When small alterations in the characteristics of the endogeneous protein are desired, substitutions are generally made in accordance with the following chart: Chart 1 30 Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gin, His Asp Glu 35 Cys Ser Gin Asn 38 WO 99/60108 PCTIUS99/10731 Glu Asp Gly Pro His Asn, Gin lie Leu, Val 5 Leu lie, Val Lys Arg, Gin, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr 10 Thr Ser Trp Tyr Tyr Trp, Phe Val lie, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart 1. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the a-helical or 0-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the 5 bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative 10 residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine. Preferred embodiments of the present invention include, but are not limited to: (1) a farm animal including cattle, sheep, pigs, horses and goats with a 1-25 base pair deletion, or a 10-25 base pair insertion of a polylinker sequence, or insertion of a reporter gene such as a luciferase gene, a P 15 galactosidase gene or a green fluorescent (GFP) protein gene in an endogenous gene or sequence encoding ornithine transcarbamylase (OTC), lactoglobulin, casein, p-casein, a-casein, K-casein, albumin, globin, immunoglobulin, igG, interleukin, a sugar transferase, integrin, a milk protein, a urine protein, a retroviral provirus, an endogenous virus, a prion, a leptin, or cystic fibrosis transmembrane regulator (CFTR); (2) a farm animal including cattle, sheep, pigs, horses and goats with an exogenous 20 gene such as a gene encoding human lysozyme, human growth hormone, human serum albumin, human globin, a human antibody (human igG), a tissue plasminogen activator, a human therapeutic protein, human lactase, a human lipase, a hormone receptor gene, a viral receptor gene, a G-protein coupled receptor gene, a drug or a human enzyme gene, including for example the human lysozyme gene, the human a-1 anti-trypsin gene, the human anti-thrombin Ill gene; (4) a farm animal including 25 cattle, sheep, pigs, horses and goats with a modified endogenous repeated (A-C)n sequence, a modified repeated (A-G)n sequence, a modified repeated (A-T)n sequence, a modified endogenous 39 WO 99/60108 PCTIUS99/10731 CFTR gene or a modified endogenous OTC gene; (5) a farm animal including cattle, sheep, pigs, horses and goats with a modified "-lactoglobulin gene or $-lactoglobulin gene does not encode any phenylalanine residues; (6) a farm animal including cattle, sheep, pigs, horses and goats with a human monoclonal antibody gene, or a gene for a human antibody that does not encode any phenylalanine 5 residues, for example inserted (or replacing) in the endogenous gene or sequence encoding an immunoglobulin, or igG; and (7) a farm animal including cattle, sheep, pigs, horses and goats with a human gene under control of its endogenous promoter, a modified endogenous regulatory element for an endogenous gene which may or may not be disrupted by an insertion sequence, a transcriptional regulation cassette ord a dimerizing sequence. Specific preferred embodiments also include, a farm 10 animal including cattle, sheep, pigs, horses and goats with an endogenous regulatory element which is disrupted by, deletion of at least one nucleotide. Additional preferred embodiments comprise a pig, monkey or cow with a 1-25 to 1-50 base pair insertion, examples of which include a hormone receptor gene, a viral receptor gene or a G-protein coupled receptor gene, or a 1-25 to 1-50 bp deletion in a sugar transferase gene including the a 15 galactosyl transferase gene (gaiT) or the fucosyl transferase gene, a BELE@ goat with a human gene, and a pig, goat, sheep or cow with a 1-25 base pair insertion or a 1-25 base pair deletion in a endogenous retroviral provirus gene such as deletion of the sequence for proviral KC. Further specific preferred embodiments include, a cow with a modified milk production gene such as, a cow with a lactase gene insertion in a milk promoter, a cow with the human lactoferrin gene replacing the bovine 20 lactoferrin gene, a monkey with a human therapeutic gene, or a human antibody gene, a cow with the human lipase gene in a milk promoter, a cow with a human gene placed in a transcription initiation site of a milk gene under the control of its endogenous promoter, a cow with a human gene placed in a transcription initiation site of a globin gene under the control of its endogenous globin gene promoter, a cow and goat with a modified urine protein gene, a mammal with a modified endogenous leptin gene, a 25 modified endogenous OTC gene, a modified endogenous CFTR gene or a modified interleukin gene. Additional preferred embodiments include an animal such as a mouse, rabbit or goat with a transcriptional regulation cassette inserted in the transcriptional start site of an integrin gene, and a mouse with a modification in the integrin gene or G-protein coupled receptor gene. The vectors containing the DNA segments of interest can be transferred into the host cell by well 30 known methods, depending on the type of cellular host. For example, micro-injection is commonly utilized for target cells, although calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection also may be used. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, and others (s, generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 35 which is incorporated herein by reference). Direct injection of DNA and/or recombinase-coated 40 WO 99/60108 PCT/US99/10731 targeting polynucleotides into target cells, such as skeletal or muscle cells also may be used (Wolff et al. (1990) Science 247: 1465, which is incorporated herein by reference). Targeting of Endogenous DNA Sequences Once made and administered to a target host cell, the compositions of the invention find use in a 5 number of applications, including the site directed modification of endogeneous sequences within any target cell, the creation of transgenic plants and animals, and the use of the compositions to do site-directed mutagenesis or modifications of target sequences. Generally, any predetermined endogenous DNA sequence, such as a gene sequence, can be altered by homologous recombination (which includes gene conversion) with an exogenous targeting 10 polynucleotides (such as a complementary pair of single-stranded targeting polynucleotides). The target polynucleotides have at least one homology clamp which substantially corresponds to or is substantially complementary to a predetermined endogenous DNA target sequence and are introduced with a recombinase (e.g., recA) into a target cell having the predetermined endogenous DNA sequence. Typically, a targeting polynucleotide (or complementary polynucleotide pair) has a 15 portion or region having a sequence that is not present in the preselected endogenous targeted sequence(s) (i.e., a nonhomologous portion or mismatch) which may be as small as a single mismatched nucleotide, several mismatches, or may span up to about several kilobases or more of nonhomologous sequence. Generally, such nonhomologous portions are flanked on each side by homology clamps, although a single flanking homology clamp may be used. Nonhomologous portions 20 are used to make insertions, deletions, and/or replacements in a predetermined endogenous targeted DNA sequence, and/or to make single or multiple nucleotide substitutions in a predetermined endogenous target DNA sequence so that the resultant recombined sequence (i.e., a targeted recombinant endogenous sequence) incorporates some or all of the sequence information of the nonhomologous portion of the targeting polynucleotide(s). Thus, the nonhomologous regions are used 25 to make variant sequences, i.e. targeted sequence modifications. Additions and deletions may be as small as 1 nucleotide or may range up to about 2 to 4 kilobases or more. In this way, site directed modifications may be done in a variety of systems for a variety of purposes. In a preferred application, a targeting polynucleotide is used to repair a mutated seguence of a structural gene by replacing it or converting it to a wild-type sequence (e.g., a sequence encoding a 30 protein with a wild-type biological activity). For example, such applications could be used to convert a sickle cell trait allele of a hemoglobin gene to an allele which encodes a hemoglobin molecule that is not susceptible to sickling, by altering the nucleotide sequence encoding the $-subunit of hemoglobin, so that the codon at position 6 of the $-subunit is converted fromVal$6-->Glu$6 (Shesely et al. (1991) op.cit.). Other genetic diseases can be corrected, either partially or totally, by replacing, inserting, 41 WO 99/60108 PCTIUS99/10731 and/or deleting sequence information in a disease allele using appropriately selected exogenous targeting polynucleotides. For example but not for limitation, the )F508 deletion in the human CFTR gene can be corrected by targeted homologous recombination employing a recA-coated targeting polynucleotide of the invention. 5 For many types of in vio gene therapy to be effective, a significant number of cells must be correctly targeted, with a minimum number of cells having an incorrectly targeted recombination event. To accomplish this objective, the combination of: (1) a targeting polynucleotide(s), (2) a recombinase (to provide enhanced efficiency and specificity of correct homologous sequence targeting), and (3) a cell uptake component (to provide enhanced cellular uptake of the targeting polynucleotide), provides a 10 means for the efficient and specific targeting of cells in vivo, making in vivo homologous sequence targeting, and gene therapy, practicable. Several disease states may be amenable to treatment or prophylaxis by targeted alteration of heptocytes i vivo by homologous gene targeting. For example and not for limitation, the following diseases, among others not listed, are expected to be amenable to targeted gene therapy: 15 hepatocellular carcinoma, HBV infection, familial hypercholesterolemia (LDL receptor defect), alcohol sensitivity (alcohol dehydrogenase and/or aldehyde dehydrogenase insufficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyrias, inherited disorders of hepatic metabolism, ornithine transcarbamylase (OTC) alleles, HPRT alleles associated with Lesch Nyhan syndrome, etc. Where targeting of hepatic cells in vivo is desired, a cell-uptake component consisting essentially of an 20 asialoglycoprotein-poly-L- lysine conjugate is preferred. The targeting complexes of the invention which may be used to target hepatocytes in vivo take advantage of the significantly increased targeting efficiency produced by association of a targeting polynucleotide with a recombinase which, when combined with a cell-targeting method such as that of W092/05250 and/or Wilson et al. (1992) J. Biol. Chem. 267:963, provide a highly efficient method for performing in vivo homologous sequence 25 targeting in cells, such as hepatocytes. In a preferred embodiment, the methods and compositions of the invention are used for gene inactivation. That is, in addition to correcting disease alleles, exogenous targeting polynucleotides can be used to inactivate, decrease or alter the biological activity of one or more genes in a cell (or transgenic nonhuman animal). This finds particular use in the generation of animal models of disease 30 states, or in the elucidation of gene function and activity, similar to Aknock out@ experiments. These techniques may be used to eliminate a biological function; for example, a galT gene (alpha galactosyl transferase genes) associated with the xenoreactivity of animal tissues in humans may be disrupted to form transgenic animals (e.g. pigs) to serve as organ transplantation sources without associated hyperacute rejection responses. Alternatively, the biological activity of the wild-type gene may be 42 WO 99/60108 PCT/US99/10731 either decreased, or the wild-type activity altered to mimic disease states. This includes genetic manipulation of non-coding gene sequences that affect the transcription of genes, including, promoters, repressors, enhancers and transcriptional activating sequences. Once the specific target genes to be modified are selected, their sequences may be scanned for 5 possible disruption sites (convenient restriction sites, for example). Plasmids are engineered to contain an appropriately sized gene sequence with a deletion or insertion in the gene of interest and at least one flanking homology clamp which substantially corresponds or is substantially complementary to an endogenous target DNA sequence. Vectors containing a targeting polynucleotide sequence are typically grown in E. coli and then isolated using standard molecular biology methods, or may be 10 synthesized as oligonucleotides. Direct targeted inactivation which does not require vectors may also be done. When using microinjection procedures it may be preferable to use a transfection technique with linearized sequences containing only modified target gene sequence and without vector or selectable sequences. The modified gene site is such that a homologous recombinant between the exogenous targeting polynucleotide and the endogenous DNA target sequence can be identified by 15 using carefully chosen primers and PCR, followed by analysis to detect if PCR products specific to the desired targeted event are present (Erlich et al., (1991) Science 252: 1643, which is incorporated herein by reference). Several studies have already used PCR to successfully identify and then clone the desired transfected cell lines (Zimmer and Gruss, (1989) Nature 338: 150; Mouellic et al., (1990) Proc. Natl. Acad. Sci. USA 87: 4712; Shesely et al., (1991) Proc. Natl. Acad. Sci. USA 88: 4294, which 20 are incorporated herein by reference). This approach is very effective when the number of cells receiving exogenous targeting polynucleotide(s) is high (i.e., with microinjection, or with liposomes) and the treated cell populations are allowed to expand to cell groups of approximately 1 x 10' cells (Capecchi, (1989) Science 244: 1288). When the target gene is not on a sex chromosome, or the cells are derived from a female, both alleles of a gene can be targeted by sequential inactivation 25 (Mortensen et al., (1991) Proc. Nat. Acad. Sci. USA 88: 7036). In addition, the methods of the present invention are useful to add exogeneous DNA sequences, such as exogeneous genes or extra copies of endogeneous genes, to an organism. As for the above techniques, this may be done for a number of reasons, including: to alleviate disease states, for example by adding one or more copies of a wild-type gene or add one or more copies of a therapeutic 30 gene; to create disease models, by adding disease genes such as oncogenes or mutated genes or even just extra copies of a wild-type gene; to add therapeutic genes and proteins, for example by adding tumor suppressor genes such as p53, Rbl, Wt1, NF1, NF2, and APC, or other therapeutic genes; to make superior transgenic animals, for example superior livestock; or to produce gene products such as proteins, for example for protein production, in any number of host cells. Suitable 35 gene products include, but are not limited to, Rad5l, alpha-antitrypsin, casein, hormones, 43 WO 99/60108 PCT/US99/10731 antithrombin 111, alpha glucosidase, collagen, proteases, viral vaccines, tissue plaminogen activator, monoclonal antibodies, Factors VIII, IX, and X, glutamic acid decarboxylase, hemoglobin, prostaglandin receptor, lactoferrin, calf intestine alkaline phosphatase, CFTR, human protein C, porcine liver esterase, urokinase, and human serum albumin. 5 Thus, in a preferred embodiment, the targeted sequence modification creates a sequence that has a biological activity or encodes a polypeptide having a biological activity. In a preferred embodiment, the polypeptide is an enzyme with enzymatic activity. In addition to fixing or creating mutations involved in disease states, a preferred embodiment utilizes the methods of the present invention to create novel genes and gene products. Thus, fully or partially 10 random alterations can be incorporated into genes to form novel genes and gene products, to produce rapidly and efficiently a number of new products which may then be screened, as will be appreciated by those in the art. In a preferred embodiment, the compositions and methods of the invention are useful in site-directed mutagenesis techniques to create any number of specific or random changes at any number of sites 15 or regions within a target sequence (either nucleic acid or protein sequence), similar to traditional site-directed mutagenesis techniques such as cassette mutagenesis and PCR mutagenesis. Thus, for example, the techniques and compositions of the invention may be used to generate site specific variants in any number of systems, including E. coli, Bacillus, Archebacteria, Thermus, yeast (Sacchromyces and Pichia), insect cells (Spodoptera, Trichoplusia, Drosophila), Xenopus, rodent cell 20 lines including CHO, NIH 3T3 and primate cell lines including COS, or human cells, including HT1080 and BT474, which are traditionally used to make variants. The techniques can be used to make specific changes, or random changes, at a particular site or sites, within a particular region or regions of the sequence, or over the entire sequence. In this and other embodiments, suitable target sequences include nucleic acid sequences encoding 25 therapeutically or commercially relevant proteins, including, but not limited to, enzymes (proteases, recombinases, lipases, kinases, carbohydrases, isomerases, peptides tautomerases, nucleases etc.), hormones, receptors, transcription factors, growth factors, antibodies, cytokines, globin genes, immunosupppressive genes, tumor suppressors, oncogenes, complement-activating genes, milk proteins (casein, "-lactalbumin, B-lactoglobulin, whey proteins, serum albumin), immunoglobulins, urine 30 proteins, milk proteins, esterases, pharmaceutical proteins and vaccines. In a preferred embodiment, the methods of the invention are used to generate pools or libraries of variant nucleic acid sequences, and cellular libraries containing the variant libraries. Thus, in this 44 WO 99/60108 PCT/US99/10731 embodiment, a plurality of targeting polynucleotides are used. The targeting polynucleotides each have at least one homology clamp that substantially corresponds to or is substantially complementary to the target sequence. Generally, the targeting polynucleotides are generated in pairs; that is, pairs are made of two single stranded targeting polynucleotides that are substantially complementary to 5 each other (i.e. a Watson strand and a Crick strand). However, as will be appreciated by those in the art, less than a one to one ratio of Watson to Crick strands may be used; for example, an excess of one of the single stranded target polynucleotides (i.e. Watson) may be used. Preferably, sufficient numbers of each of Watson and Crick strands are used to allow the majority of the targeting polynucleotides to form double D-loops, which are preferred over single D-loops, as outlined above. In 10 addition, the pairs need not have perfect complementarity; for example, an excess of one of the single stranded target polynucleotides (i.e. Watson), which may or may not contain mismatches, may be paired to a large number of variant Crick strands, etc. Due to the random nature of the pairing, one or both of any particular pair of single-stranded targeting polynucleotides may not contain any mismatches. However, generally, at least one of the strands will contain at least one mismatch. 15 The plurality of pairs preferably comprise a pool or library of mismatches. The size of the library will depend on the number of residues to be mutagenized, as will be appreciated by those in the art. Generally, a library in this instance preferably comprises at least 40% different mismatches, with at least 30% mismatches being preferred and at least 10% being particularly preferred. That is, the plurality of pairs comprise a pool of random and preferably degenerate mismatches over some regions 20 or all of the entire targeting sequence. As outlined herein, Amismatches@ include substitutions, insertions and deletions. Thus, for example, a pool of degenerate variant targeting polynucleotides covering some, or preferably all, possible mismatches over some region are generated, as outlined above, using techniques well known in the art. Preferably, but not required, the variant targeting polynucleotides each comprise only one or a few mismatches (less than 10), to allow complete 25 multiple randomization, as outlined below. As will be appreciated by those in the art, the introduction of a pool of variant targeting polynucleotides (in combination with recombinase) to a target sequence, either in vitro to an extrachromosomal sequence or in vivo to a chromosomal or extrachromosomal sequence, can result in a large number of homologous recombination reactions occuring over time. That is, any number of homologous 30 recombination reactions can occur on a single target sequence, to generate a wide variety of single and multiple mismatches within a single target sequence, and a library of such variant target sequences, most of which will contain mismatches and be different from other members of the library. This thus works to generate a library of mismatches. 45 WO 99/60108 PCTIUS99/10731 In a preferred embodiment, the variant targeting polynucleotides are made to a particular region or domain of a sequence (i.e. a nucleotide sequence that encodes a particular protein domain). For example, it may be desirable to generate a library of all possible variants of a binding domain of a protein, without affecting a different biologically functional domain, etc. Thus, the methods of the 5 present invention find particular use in generating a large number of different variants within a particular region of a sequence, similar to cassette mutagenesis but not limited by sequence length. In addition, two or more regions may also be altered simultaneously using these techniques. Suitable domains include, but are not limited to, kinase domains, nucleotide-binding sites, DNA binding sites, signaling domains, structural domains, receptor binding domains, transcriptional activating regions, 10 promoters, origins, active enzyme domains, dimerizing domains, leader sequences, terminators, localization signal domains, and, in immunoglobulin genes, the complementaity determining regions (CDR), Fc, VH and V, In a preferred embodiment, the variant targeting polynucleotides are made to the entire target sequence. In this way, a large number of single and multiple mismatches may be made in an entire 15 sequence. Thus for example, the methods of the invention may be used to create superior recombinant reporter genes such as lacZ, luiciferase and green fluoroscent protein (GFP); superior antibiotic and drug resistance genes; superior recombinase genes; superior recombinant vectors; and other superior recombinant genes and proteins, including peptides, immunoglobulins, vaccines or other proteins with 20 therapeutic value. For example, targeting polynucleotides containing any number of alterations may be made to one or more functional or structural domains of a protein, and then the products of homologous recombination evaluated. Once made and administered to target cells, the target cells may be screened to identify a cell that contains the targeted sequence modification. This will be done in any number of ways, and will 25 depend on the target gene and targeting polynucleotides, as will be appreciated by those in the art. The screen may be based on phenotypic, biochemical, genotypic, or other functional changes, depending on the target sequence. In an additional embodiment, as will be appreciated by those in the art, selectable markers or marker sequences may be included in the targeting polynucleotides to facilitate later identification. 30 In a preferred embodiment, kits containing the compositions of the invention are provided. The kits include the compositions, particularly those of libraries or pools of degenerate cssDNA probes, along with any number of reagents or buffers, including recombinases, buffers, ATP, etc. 46 WO 99/60108 PCTIUS99/10731 The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the invention in any manner. All references cited herein are expressly incorporated by reference. EXPERIMENTAL EXAMPLES 5 EXAMPLE 1 Homoloqous Targetinq of recA-Coated Chemically-Modified Polynucleotides in Cells Homologously targeted exogenous targeting polynucleotides specifically target human DNA sequences in intact nuclei of metabolically active cells. RecA-coated complementary exogenous targeting polynucleotides were introduced into metabolically active human cells encapsulated in 10 agarose microbeads and permeabilized to permit entry of DNA/protein complexes using the Jackson Cook method (Cook, P.R. (1984) EMBO J. 3: 1837; Jackson and Cook (1985) EMBO J. 4: 919; Jackson and Cook (1985) EMBO J. 4: 913; Jackson and Cook (1986) J. Mol. Biol. 192: 65; Jackson et al. (1988) J. Cell. Sci. 90: 365, which are incorporated herein by reference). These experiments were designed to specifically target homologous DNA sequences with recA protein in intact nuclei of 15 metabolically active human HEp-2 cells. Jackson and Cook previously demonstrated that the nuclear membranes of human or other cells may be permeabilized without loss of metabolic function when the cells are first encapsulated in a gel of agarose microbeads. The agarose microbead coat contains the cell constituents and preserves native conformation of chromosomal DNA, while permitting diffusion of macromolecules into and out of the 20 cell compartment. Wittig et al.(1991) Proc. NatI. Acad. Sci. (U.S.A.), 8: 2259, which is incorporated herein by reference, demonstrated that monoclonal antibodies directed against left-handed Z-DNA could be diffused into these agarose-embedded cells, and that the antibodies were specifically targeted to chromosomal sequences and conformations. In a similar manner, we incubated biotin- or FITC-labeled complementary DNA targeting polynucleotides coated with recA with agarose-coated cell 25 nuclei and verified the correct homologous targeting of the exogenous targeting polynucleotides to specific predetermined human DNA sequences in cell nuclei of metabolically active cells. RecA-mediated homologous gene targeting with complementary oligonucleotides in intact human cell nuclei was verified directly by homologous targeting using targeting polynucleotides that were biotinylated. These were subsequently labeled with a fluorescent reporter compound to verify 30 homologous pairing at specific locations having the predetermined sequence(s). RecA-coated targeting polynucleotides for human chromosome 1 pericentrometric alpha-satellite DNA sequences 47 WO 99/60108 PCT/US99/10731 were specifically targeted to chromosome 1 centromere sequences in living human cell nuclei that were permeabilized and suspended in agarose. In these experiments, recA-coated biotinylated exogenous targeting polynucleotides containing homologous sequences to human chromosome 1 alpha satellite DNA were incubated with human 5 HEp-2 cells. The cells were embedded in agarose, then treated with standard buffers (according to Jackson and Cook, op.cit.) to remove the cytoplasmic membrane and cytoplasm immediately before the addition of targeting polynucleotide coated with recA protein. The experiments were performed with the following results: First, in order to test protocols to be used in nuclear encapsulation, freshly trypsinized growing human 10 HEp-2 tumor cells were suspended in complete DMEM encapsulated in a mixture of agarose (2.5%, Fisher-Biotech) and complete DMEM media adapting the protocols of Nilsson et al., 1983, so that the final agarose concentration was 0.5% (4 volumes cells in suspension with 1 volume 2.5% agarose), and the final cell concentration range was approximately 2.4 x 107 to 8 x 105. The encapsulated cells in agarose "beads@ were placed in petri dishes to which DMEM complete media was added and were 15 allowed to grow for 24 hr in an incubator at 37'C , 7% CO 2 . At 24 hr, the cells were clearly growing and multiplying and thus were alive and metabolically active. An aliquot of agarose containing cells (in beads in DMEM medium) was treated to remove the cytoplasmic membrane and cytoplasm by addition of ice-cold sterile PBS, New Buffer (Jackson et al. (1988) op.cit; 130 mM KC1, 10 mM Na 2

HPO

4 , 1 mM MgC1 2 , 1 mM Na 2 ATP, and 1 mM dithithreitol, pH 20 7.4 ), New Buffer with 0.5% Triton-X 100, New Buffer with 0.2% BSA, then was centrifuged at low speed using protocols developed by Jackson and Cook, 1985 and 1986 op.cit.; Wittig et al. (1989). Cell. Biol. 108: 755; Wittig et al. (1991) op.cit.) who have shown that this treatment allows the nuclear membrane to remain morphologically intact. The nuclei are metabolically active as shown by a DNA synthesis rate of 85 to 90% compared with that of untreated control cells. 25 Cytoplasm was effectively removed by the above treatment, and the encapsulated nuclei were intact as demonstrated by their morphology and exclusion of 0.4% trypan blue. Nuclei in agarose were returned to the humidified CO 2 incubator at 37 0 C for 24 hr and remained metabolically active. We observed that sterile mineral oil used in the emulsification process was difficult to remove entirely and interfered with the microscopic visualization of suspended nuclei. Therefore, the cell-agarose 30 suspension process was simplified. In subsequent experiments cells were gently vortexed with melted (39 0 C) agarose, then the agarose-cell mixture was sterilely minced before New Buffer treatments. 48 WO 99/60108 PCT/US99/10731 This simpler process, eliminating the oil step, makes it easier to visualize the cells and chromosomes at the completion of reactions. After mincing of the agar and New Buffer treatments of the cells, the above protocols were used to homologously target endogenous DNA sequences in encapsulated nuclei as follows: 16.5 pl recA 5 coated (or non-recA-coated control) nick-translated DNA (labeled with biotin-14-dATP) targeting polynucleotide was prepared and bound under standard native recA protocols (se U.S.S.N. 07/755,462 and 07/910,791). Minced agarose fragments were centrifuged and New Buffer supernatant removed. The fragments were resuspended in 1 X AC buffer in a 1.5-ml Eppendorf tube, then centrifuged for removal of the buffer (leaving an estimated 50 to 75 pl of buffer), and prepared 10 targeting polynucleotide was mixed with the fragments of agarose-containing nuclei. Reactions were incubated in a 370C water bath for 2 to 4 hr, then washed, incubated in standard preblock solution, then in preblock supplement with 10 pg/ml FITC-avidin (Vector, DCS grade), and again washed. Experimental results were analyzed by placing a minute amount of a reaction with 3 to 4 pi antifade on a slide with a slide cover and viewing it by using the Zeiss CLSM-1 0 confocal laser scanning 15 microscope (CLSM). Completed reactions were also stored refrigerated for later examination. In the first in vivo experiment, metabolically active HEp-2 cells suspended in 1 x PBS were encapsulated in agarose by gentle vortexing, treated using New Buffer protocols, then incubated for 3 hr 15 min with 100 ng of recA-coated targeting polynucleotide specific for Chromosome 1 alpha satellite DNA biotinylated with bio-14-dATP by nick translation (BRL, Nick Translation System) using 20 pUC 1.77 plasmid DNA (a 1.77 kb long EcoRI fragment of human DNA in the vector pUC9; Cooke et al. (1979) Nucleic Acids Res. 6: 3177; Emmerich et al. (1989) Exp. Cell. Res. 181: 126). We observed specific targeting by the alpha-satellite targeting polynucleotide to pericentromeric chromosome 1 targets in intact nuclei of metabolically active cells. The signals were essentially identical to those using the same targeting polynucleotide with methanol (or ethanol) fixed HEp-2 cell targets in 25 suspension. Figure 1 shows specific targeting signals in several metabolically active cells from this experiment. In the second in vivo experiment, cells suspended in incomplete DMEM media instead of 1 x PBS were encapsulated in agarose and treated with 62.5 ng of the same targeting polynucleotide used in the first experiment described above and 62.5 ng of a freshly biotinylated targeting polynucleotide 30 prepared under the same protocols. In this experiment, the minced agarose fragments were not resuspended in 1 x AC buffer before addition of targeting polynucleotide and some nuclei disintegrated, especially with subsequent centrifugation. The results show that in the nuclei that remained intact, the targeting polynucleotides coated with recA specifically targeted predetermined 49 WO 99/60108 PCT/US99/10731 human DNA targets. In contrast, targeting polynucleotides in control reactions without recA did not target the human DNA sequences. Thus, the recA-coated targeting polynucleorides were targeted to the repetitive alpha satellite sequences of chromosome 1. This result showed DNA targeting in intact nuclei to specific human 5 chromosome 1 sequences (data not shown). In the third experiment, cells were suspended in 1 x PBS or in incomplete DMEM media before vortexing with agarose and were tested using 62.5 ng of targeting polynucleotide in reactions with and without recA protein. In addition, the reactions were divided in half and washed and FITC-avidin treated in either buffer adjusted to pH 7 or pH 7.4. Cells were incubated with the recA coated targeting 10 polynucleotide for 3 hr 25 min. Live nuclei treated with targeting polynucleotide alone without recA showed no signals. In the recA-treated reactions, relatively weaker signals were observed in nuclei incubated in 1 x PBS, whereas very strong specific signals were present in nuclei that had been incubated in incomplete DMEM. There was clearly significantly more signal present in nuclei that were washed and treated with FITC-avidin at pH 7.4 compared with nuclei incubated at pH 7.0. Figure 4 15 shows nuclei that were treated with recA coated targeting polynucleotides and incubated at both pH 7.4 and 7.0. In a fourth experiment, HEp-2 cells were embedded in agarose prepared with I x PBS, New Buffer treated, then treated with 100 ng of biotinylated targeting polynucleotide complementary to chromosome 1 alpha-satellite DNA. Controls in this experiment also included reactions without recA 20 protein and additional control reactions supplemented with an identical amount of BSA protein to replace the recA protein. Additionally, cells were also embedded in agarose prepared with I x AC buffer. Examples of specific targeting to endogenous target sequences were recorded. In a fourth experiment, we directly determined if the embedded nuclei under the conditions used above were metabolically active. The nuclei in agarose were incubated with bio-21-rUTP in complete 25 medium, then incubated for 2 days in the humidified CO 2 atmosphere. After 2 days at 37 0 C, the cells were examined. Bio-21-rUTP was incorporated in RNA and incubated with FITC-streptavidin. FITC was specifically associated with nucleoli indicative of ribosomal RNA biosynthesis, thus directly showing metabolic activity in these human cells. Similar results were obtained using DNA precursors to measure DNA synthesis. In this experiment it was clear that the majority of nuclei in the PBS 30 agarose reaction had condensed chromosomes. There was nuclear activity in a number of these nuclei also, indicative of full metabolic viability, which was also shown in the AC buffer-treated cells. A fifth experiment was performed using, again, HEp-2 cells embedded in agarose. Final concentration of the cells in agarose was 3.7 x 10 6 /ml. The cells were suspended in 1 x PBS prior to combining with 50 WO 99/60108 PCT/US99/10731 agarose. The final agarose concentration was 0.5%. There were two reactions, one in which recA was used to coat targeting polynucleotide, the second in which recA protein was replaced by BSA at the same protein concentration followed by New Buffer treatments to remove the cytoplasm. The nuclei in agarose were incubated for 3 hr with targeting polynucleotide, then processed for detection of 5 correctly targeted polynucleotide using the protocols describe previously. FITC-avidin was used to visualize the biotinylated targeting polynucleotide at a concentration of 20 pg/ml. Results showed that cells with the recA-coated complementary targeting polynucleotide displayed specific signals in 25% or more of the intact nuclei. In contrast, the BSA-treated controls (without RecA) did not show any signal. Cells in agarose from this experiment were further incubated at 370C in the C02 incubator in complete 10 medium. At 22 hr, these cells were metabolically active. Chromosomes were condensed, and a number of nuclei were in the process of dividing. In these experiments, a significant number of the cells incubated with recA-coated complementary targeting polynucleotides showed specific signal, whereas 0% of the cells incubated with targeting polynucleotide alone showed specific signal. In summary, recA-coated biotinylated targeting polynucleotides for human chromosome 1 alpha 15 satellite DNA were specifically targeted to human HEp-2 epithelial carcinoma chromosomal DNA in intact cell nuclei of metabolically active cells that had been suspended in agarose, then treated with buffers and recA-coated targeting polynucleotides under suitable reaction conditions (supra and U.S.S.N. 07/755,462; U.S.S.N. 07/755,462; and U.S.S.N. 07/520,321, incorporated herein by reference). Specific binding by the recA-coated targeting polynucleocide to chromatin alpha-satellite 20 DNA was observed only in the agarose embedded nuclei which were incubated with recA-coated targeting polynucleotides. Control nuclei incubated with targeting polynucleotides in the absence of recA and/or with nonspecific protein exhibited no signal. Targeting of Human p53 Gene We performed recA-mediated homologous targeting of biotinylated targeting polynucleotides that were 25 homologous to the human p53 tumor suppressor gene, and compared the results to targeting of alpha satellite DNA sequences in human chromosome 1. In these experiments, exponentially growing cells were trypsinized, washed, suspended in incomplete medium and encapsulated in ag.arose. The agarose was minced into pieces with a razor blade and the encapsulated cells were treated with New Buffer. A sample from each group was removed to verify that nuclei were intact. 30 Nuclei were washed in 1 x AC buffer and incubated with recA-coated complementary single-stranded DNA oligonucleotides (i.e., exogenous targeting polynucleotides) for 3.5 hours at 37 0 C. The alpha satellite DNA targeting polynucleotides for chromosome 1 were previously described and were nick 51 WO 99/60108 PCT/US99/10731 translated with biotinylated deoxyribonucleotides (bio-14-dATP). The p53 tumor suppressor gene polynucleotide was obtained from Oncor (209 Perry Parkway, Gaithersburg, MD 20877) and is a 1.2 kilobase cDNA fragment from a wild-type human p53 gene (Fields and Jang, (1990) Science 242: 1046; Miller et al. (1986) Nature 319: 783; Zakut-Houre et al. (1985) EMBO J. 4: 1251). The 1.2 5 kilobase human p53 DNA was nick-translated with biotinylated deoxyribonucleotides and yielded a population of biotinylated targeting polynucleotides having a size range (about 100 to 600 nucleotides) similar to that obtained for the human chromosome 1 alpha satellite targeting polynucleotides. The targeting polynucleotides were separately incubated with encapsulated cells. Following incubation 3 washes of 1.75 x SSC were done, and sampled nuclei were verified as intact after the washing step. 10 After washing, the targeted encapsulated cell nuclei were incubated in preblock and FITC-avidin was added to preblock buffer to a final concentration of 20 pg/mI for 15 minutes in the dark. The targeted encapsulated cell nuclei were washed sequentially in 4 x SSC, 4 x SSC with 0.1% Triton X-100, and then 4 x SSC. Samples of nuclei were again taken and used to verify that the targeted nuclei were metabolically active. Microscopic examination showed that metabolically active cells contained 15 specific FITC-targeting polynucleotide: targeted endogenous sequence complexes (shown in Figure 2). The p53 targeting polynucleotides were specifically targeted to human chromosome 17, the location of the endogenous human p53 gene sequences, indicating specific pairing of a targeting polynucleotide to a unique endogenous DNA target sequence. The human chromosome 1 alpha satellite DNA was also specifically targeted to the chromosome 1 pericentromeric satellite sequences. 20 The experiments validated a highly specific DNA targeting technique for human or other cells as evidenced by homologous sequence targeting techniques in metabolically active cells. The targeting technique employs the unique properties of recA-mediated DNA sequence targeting with single stranded (complementary) short targeting polynucleotides. Native intact nuclei were incubated with labeled, heat-denatured targeting polynucleotides coated with recA protein. The DNA hybridized to 25 the predetermined targeted homologous sequences. In these experiments, the targeting polynucleotides formed paired complexes with specific gene sequences within metabolically active cell nuclei. This in vivo targeting by recA-mediated homologous targeting polynucleotides shows the targeting specificity and therapeutic potential for this new in vivo methodology. Application of recA or other recombinase-mediated targeting of (complementary) ssDNA or denatured dsDNA targeting 30 polynucleotides to predetermined endogenous DNA targets is important for gene entry, gene knockout, gene replacement, and gene mutation or correction. EXAMPLE 2 Correcting a Mutant Gene to Produce a Functional Gene Product 52 WO 99/60108 PCT/US99/10731 Homologously targeted complementary DNA oligonucleotides were used to correct 11 bp insertion mutations in vector genes and restore vector gene expression and vector protein function in microinjeate mammalian cells. Experimentswere designed to testwhether homologously targeted complementary 276-bp oligonucleotil 5 targeting polynucleotides could correct an 1 1-bp insertion mutation in the lacZ gene of a mammaliiNA vector, which encoded a nonfunctional $-galactosidase, so that a corrected lacZ gene encoded and expressed a functional enzyme. Functional enzyme ($-galactosidase) was detected by an X-gal assay that turns cells expressing a revertant (i.e., corrected) lacZ gene a blue color. NIH3T3 cells microinjected with the mutant test vector bearing an 11 basepair insertion indliacZ coding 10 sequence do not produce any detectable functional $-galactosidase enzyme. In contrast, cells microinjected with the wild type test vector do produce functional enzyme. We obtained the functional lac plasmid pMC11acpA for use as a positive control for expression of $ galactosidase. pMC11acXpA is the target test mutant plasmid (shown in Figure 3). It is identical to pMC1 acpA (shown in Figure4) but has a 11 -bp Xbal linker insertional mutation. This plasmid does not 15 express $-galactosidase activity in rouse NIH3T3 cells when introduced by electroporation. It does not produce blue color in the presence of X-gal indicative of $-galactosidase production following vector micro-injection. NEgative controls with mock or noninjected cells we also done. Using these conditions and NIH3T3 cells have no detectable background blue staining. The plasmid pMC1 lacpA (8.4 kb) contains the strong polyoma virus promoter of transcription plus ATG 20 placed in front of the lacZ gene. The polyadenylation signal from SV40 virus was placed in back of the lacZ gene. The plasmid vector was plB130 from IBI (New Haven, CT). The mutant vector pMC11acpA has a 11-bp inserton in the Xbal site consisting of the inserted sequence CTCTAGACGCG (see Figure 5). In several control micro-injection experiments using pMC1 IacXpA weonsistently failed to detect any ble 25 microinjected cells. In contrast, in various experiments monitored early after microinjection apipimately 9 to 13% of the NIH3T3 cells injected with pMC1IacpA ISIA expressed $-galactosidase as evidenced by their blue color. No cells microinjected with injection buffer alone omock injected were observed as blue We synthesized two 20-bp primers (PCR" and PCR$) for producing a 276-bp PCR product (see Figure 5) from the wild-type lacZ sequence for use as targeting polynucleotides. We chose this 276-bp front 30 to span the 11 bp insertion mutation as a nonhomologous sequence. The 276-bp DNA oligonucleotide was separated by gel electrophoresis and electroeluted from agarose, ethanol precipitated, and its concentration determined by absorbance at 260 nm. The 276-bp fragment was 5' end-labeled with 1 2 P 53 WO 99/60108 PCT/US99/10731 and specifically D-looped with the pMc1lacXpA or pMC1lacpA plasmid DNA using recA as shown by agarose gel electrophoresis. Experiments were designed to test for $-galactoside production in cells microinjected with pMC1 lacXpA vectors with targeting polynucleotide-target complexes using complementary 276-bp oligonucleotide 5 targeting polynucleotide treated with recA. The 276-mer targets polynucleotides in I X TE buffer: were denatured by heating at 1000C for 5 min and immediately quenched in an ice bath for 1 min. The DN A solution was collected at 4 0 C by centrifugation. RecA-mediated targeting polynucleotide reactions containing a final volumeof 10 pl were assembled using 1.0 pl 10 x AC buffer, 1.5 p1 16 mM ATP(S, 3.8 pl dd H20, 1.2 pl recA protein solution (13 pg/pl), and 2.5 pl of a 30 pg/mI stock of headenatured 276-bp 10 targeting polynucleotide. The recA protein was allowedct coat the DNA for 10 min at 37 0 C. Next, 1.0 pl of 10 x AC buffer, 10 pl of 0.2 M magnesium acetate, 1.3 pl of pMCIlacXpA (1.0 pg/pl), and 6.7 pl of dd

H

2 0 was added to a final volume of 20 pl. Control reactions werperformed without added recA protein. NIH3T3 cells were capillary needle microinjected with targeting polynucleotide-target DNA mixtures loade in glass pipettes freshly pulled into microneedles using a Sutter instruments microprocessor controlled 15 apparatus. An ECET Eppendorf microinjection pump ad computerized micromanipulator were used for computer-assisted microinjection using an Olympus IMT-2 inverted microscope. Cells were carefully microinjected under controlled pressure and time. NIH3T3 cells injected with pMC1lacpA showed approximately 9% of the injected cells were blue. None (0%) of the celisjected with pMC11acXpA DNA in reactions containing the 271 bp oligonucleotide but without recA protein showed a blue color. In mare 20 contrast, approximately 3.6% of the cells microinjected with the recA-coated 271-bp targeting polynucleotide targeted to-the pMC1IacXpA target hybd were blue (Figure 6), indicating that the mutant pMC1acXpAgene can be targeted and corrected by the 271-bp oligonucleotide, whichas been targeted with recA-coated targeting polynuaotides. In summary, these measurements show that the 11 bp Xba I insertion mutation can be corrected with the recA-mediated targeted corrected in vivo but not with the 25 271-bp oligonucleotide alone. Notethat the in situ identification of 3T3 cells expressing $-galactosidase was performed following incubation with X-gal (5-bromo-4-chloro-3-indolyl-$- galactopyranoside) (Sigma) as described by Fische- et al. (1988) Nature 332: 853; Price et al. (1987) Proc. NatI. Acad. Sci. (U.S.A.) 84: 156; Lim and Chae (1989) BioTechniques 7: 576. EXAMPLE 3 30 Correctinq a Human CFTR Disease Allele Homologously targeted complementary DNAoligonucleotides were used to correct a naturally occurring 3 bp deletion mutation in a human CFTR allele and restore expression of a functional CFTR protein in targeted mammalian cells. 54 WO 99/60108 PCT/US99/10731 A major goal of cystic fibrosis (CF) gene therapy is the correction of mutant portions of the CF transmembrane conductance regulator (CFTR) gene by replacement with wild-type DNA sequences to restore the normal CFTR protein and iortransport function. Targeting polynucleotides that were coated with recA protein were introduced into transformed CF airway epithelial cells, homozygous for botheles 5 AF508 CFTR gene mutation, by either intranuclear microinjection, elealporation, or by transfection with a protein-DNA-lipid complex. Isolation and characterization of the CFTR gene (Rommens et al. (1989) Science 245: 1059; Riordan et al. (1989) Science 245: 1066, incorporated herein by reference) has been crucial for understanding the biochemical mechanism(s) underlying CF pathology. The most common mutation associated with CF, 10 a 3-base-pair, in-frame deletion eliminating a phenylalanine at amino acidqsition 508 (AF508) of CFTR, has been found in about 70% of all CF chromosomes (Kerem et al. (1989) Science 245: 1073; Kerem et al. (1990) Proc. Nati. Acad. Sci. (U.S.A.)87: 8447). Correction ofAF508 and other CFTR DNA mutations lies at the basis of DNA gene therapy for CF disease. Elimination of the cAMP-dependent C1 ion transport defect associated with CFTR gene mutations has been accomplished through the introduction 15 of the transcribed portion of wild-type CFTR clB1A into CF epithelial cells (Rich et al. (1990) Nature 347: 358; Drumm et al. (1990) Cell 62: 1227). An immortalized CF tracheobronchial epithelial humarcell line, ECFTE29o-, is homozygous for thenF508 mutation (Kunzelmann et al. (1993) Am. J. Respir. Cell. Mol. Biol., 8:522). These cells are useful as targets for homologous recombination analysis, because they contain the same 3 basepair deletion in 20 CFTR allele on all copies of chomosome 7. Replacement of the AF508 allele with wild-type CFTR DNA in indicated only when homologous recombination has occurred. The 491 bp region of the CFTR gene spanning exon 11 and containing 3' and 5' flanking intron sequences was selected from sequence data published previously (Zielenski et al. (1991) Genomics 10: 214, incorporated herein by reference) and used as a targeting polynucleotide. ThEDNA fragment was PCR amplified in preparative quantities and 25 then denatured for introduction into cells as recA-coated complementary ssDNA (or dsDNA). Exponentially growing cdls were transfected by intranuclear microinjection and were propagated on the same petri dishes in which they wee microinjected. Cells outside the microinjected area were removed by scraping with a rubber policeman. Exponentially growing cells were typsinized and washed before electroporation. Cells transfected with protein-DNA-lipicbomplexes were grown to approximately 70-80% 30 confluence before transfection. The 491 bp fragment was generated by PCR amplification from the T6/20 plasmid (Rommenet al. (1989) op.cit., incorporated herein by reference) ad verified by restriction enzyme mapping and propagated as described previously. After digestion with EcoRI and HindlIl, a 860 bp insert was isolated following electrophoresis in 0.8% SeaPlaque agarose gel. The 860 bp fragmt contained CFTR exon 10, as well 55 WO 99/60108 PCT/US99/10731 as 5' and 3' intron sequences, as defined by the restriction enzyme cleavagstes (Zielenski et al. (1991) op.cit.). A 50 ng aliquot of the fragment was amplified by PCR using primers CF1 and CF5 (Table 1) to generate a 491 bp fragment. The conditions for amplification were denaturation, 940C for 1 annealing, 530C for 30 sec; extension, 720C for 30 sec witle 4 sec/cycle increase in the extension time for 40 cycles 5 The fragment size was confirmed by electrophoresis on a 1% agarose gel, then amplified in bulk in 20 separate PCR amplifications, each containing 50 ng of target DNA. The 491 bp PCR products were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) extraction and precipice with ethanol. DNA precipitates were collected by centrifugabn in an Eppendorf microcentrifuge and resuspended at a final concentration of 1 mg/ml. The 491 bp fragment contained exon 10 (193 bp), as well as 5'(163 bp) and 10 3' (135 bp) flanking intron sequences, as defined by primers CF1 and CF5. The 491 nucleotide fragments were coa-d with recA protein using the reaction buffer of Cheng (Cheng, et al. (1988) J. Biol. Chem. 263:15110, incorporated herein by reference). Typically, the 491 bp DNA fragment (5pg) was denatured at 95*C for 10 rim, then added to a 63 pl of coating buffer containing 200 pg of recA protein, 4.8 mM ATP(S, and 1.7 pl reaction buffer (100 mM Tris-Ac, pH 7.5 at 37*C; 10 m M 15 dithiothreitol; 500 mM NaOAc, 20 mM MgOAc, 50 percent glycerol) and incubated for 10 min at 370C. Next, the MgOAc concentration was increased to a final concentration of about 22 mM by addition of 7 pl of 200 mM MgOAc. Under these conditions, the 491 nucleotide fragment was coated witbrA protein at a molar ratio of 3 bases per 1 recA molecule. After coating theligments were immediately placed on ice at 40C until transfection (10 min to 1 hr). 20 Microinjection, when used, was performed with an Eppendorf 5242 microinjection pump fitted to an Eppendorf 5170 micromanipulator using borosilicate pipes (Brunswick, 1.2 OD x 1.91D) fabricated into a microneedle with a Sutter Instruments (P-87) micropipette puller. The micropipettes were filled by capillary force from the opposite side of the needle. Approximately 100 pipettes were used for injecting 4000 cells. Cells were injected with approximately 1,000-10,000 fragments per cell by intranuclear 25 injection witi 120 hPa for 0.1-0.3 s at a volume of 1-10 fl/nucleus. Microinjected cells were viewed with an Olympus IMT-2 inverted microscope during the injection. The area of the petri dish containing injed cells was marked with 2 to 5 mm diameter rings. Needlenicroinjection was performed in cells grown on 10 separate 60 mm petri dishes. Cells were injected at room temperature in culture medium after two washes in phosphate buffered saline (PBS). After microinjection, noninjected cells in the culture were 30 removed by scraping. Injected cells were grown at 37*C in a humidified incubator at 7 days and then harvested for DNA and RNA. Electroporation experiments were performed using recA-coated 491-mer ssDNA as described above. Approximately 1 x 1 (Y exponentially growing cells were suspended in 4 00pl of coating buffer with 5 pg of recA coated-DNA. The cell suspension wa pre-incubated on ice for 10 min and electroporated at room 56 WO 99/60108 PCT/US99/10731 temperature with 400 V and 400pF in a BTX 300 electroporator (BTX Corporation, San Diego, CA). Afte electroporation, cells were incubted on ice for an additional 10 min, diluted in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and IDpg/ml streptomycin, 100 U/ml penicillin (Cozens et al. (1992)Proc. Natl. Acad. Sci. (U.S.A.)89: 5171; Gruenert et al. (1988) Proc. NatL. 5 Acad. Sci. (U.S.A.) 85: 5951; Kunzelmann, (1992) op.cit.), and then seeded in T75 flasks. Under these conditions of elecroporation, approximately 30-50% of the cells survive. Cells we cultured for 507 days at 370C and then harvested for DNA and RNA. Protein DNA-lipid complexes (liposomes) were prepared. Briefly, dioleoylphosphatidyl-ethanolamine (PtdEtn, DOPE) was used for preparing liposomes by $/ing 4 pM solutions of the lipid under nitrogen at 10 room temperature. The lipid film was rehydrated with 4 ml of 30 mM Tris-HC1 buffer (pH 9), then sonicated for 15 minutes under an atmosphere or argon. The protein-DNA complex was prepared in polystyrene tubes by diluting 20 pg of recA-coated 491-base DNA in 30 mM Tris-HC1, (pH 9) buffer. Gramicidin S protein (GmS) was also diluted with 30 mM Tris HC1 (pH 9) to a final concentration of 2 mg/ml from a 20 mg/ml stock solution prepared in dimethyl sulfoxide. The protein (40 pg) was added to 15 the DNA and rapidly mixed. Next, 175 pl of the liposome solution (175 nmoles dpld) were added to the peptide DNA mixture. Genomic DNA was isolated and purified from cells as described in Maniatisp.ci. to test for homologous DNA recombination. Cellular DNA was firsPCR-amplified with primers CF1 and CF6 (Table 1). CF1 is within the region of homologydefined at the 5' end of the 491 bp CFTR fragment CF6 is outside the regiw 20 of homology at the 3' end of this fragment. The conditions for the PCR amplification were as follows: CF1/CF6; 684/687 bp fragment; primers, 0. 5 pM; DNA, 1-2 pg; denaturation; 94 0 C for 1 min; annealing; 53 0 C for 45 s; extension; 720C for 90 s wit h a 4-s/cycle increase in extension time for 40 cycles; Mg1 2 1.5 mM. DNA fragments were separated by agarose electrophoresis and visualized by staining with ethidium bromide, then transferred to Gene 25 Screen Plus filters (DuPont). The DNA was then hybridized with the allele-specific normal CFIlP-end labeled DNA probe defined by oligo N as described by Cozens et al. (1992) op.cit.; Kunzelmann (1992) op.cit., incorporated herein by reference. The presence of wild-type (WT) sequences was determined autoradiographically by hybridization with the radiolabeled DNA probe. Homologous recombination was verified ire second round of PCR DNA amplification using the 687/684 30 bp fragment as a DNA template for amplification. The primers used in this allele-specific reaction were CFI and the oligo N or oligoAF. The size of the DNA fragments was 300 bp (oligo N) or 299 bp (oligAF). 57 WO 99/60108 PCT/US99/10731 The conditions for the reaction wereas follows: CF1/oligo N/AF; 300/299 bp fragment; primers, 0.5 pM; DNA, 1-2 pg; denaturation, 95*C for 45s; annealing, 510C for 30s; extension, 72 0 C for 30 s with a 3-s/cycl increase in extension time for 40 cycles; Mg+ 2 , 1.5 mM. In DNA from transfected ECFTE29o- cells, amplified with the CF1/oligo N primers, a PCR product was detected only if the wild-type CFTR sequence 5 were present. Amplification with the CFl/oligcAF gives a PCR DNA productof DNA targets purified from transfected and nontransfected 3CFTE29o- cells but not for DNA targetsolated from control normal ceil (16HBE140-). The presence of wild-type CFTR sequences in the amplified DNA fragments was also determined autoradiographically after hybridization with 32 P-5-end-labeled oligo N as probe. Cytoplasmic RNA was isolated and denatured at 950C for 2 min, then reveetranscribed using the DNA 10 polymerase provided in a PCR RNA Gene Amp kit according to manufacturer's instructions (Perkin Elmer/Cetus). First strand cDNA was amplified by using primer CF17 at the 5' end of exon 9 and the allele-specific oligo N or oligoAF primers. The length of the PCR fragments is 322 bp (CF17/oligo Jnd 321 bp (CF17/oligo AF). The conditions for PCR amplification are CF17/oligo N/AF, 322/321 bp fragment; primers, 1 pM; 15 denaturation, 940C for1 min; annealing, 51*C for 30s; extension, 72*C for 20s with a 4-s/cycle increase in extension time for 40 cycles; Mg.

2 , 0.8 mM. DNA fragments were visualized after electrophoresis on ethidium bromide-stained 1% agarose gels. In addition to the allele-specific PCR amplification of first-strand cDNA, Southern hybridization was performed as described above. Fragments were transferred to Gene Screen Plus filtes then hybridized with allele-specific oligo N probe under the same 20 conditions used for the Southern analysis of the genomic DNA (Kureimann et al. (1992) op&jt.; Cozens et al. (1992) op.cit.). The presence of wild-type CFTR RNA was confirmed by hybridization and autoradiography of RNA extracted from normal (16HBE14o-) control DNA and in DNA of transfected 3CFTE29o-cells. Hybridization was performed as described previously (Cozens et al. (1992ip.cit.). DNA fragments were 25 separated by agarose gel electrophoresis. DNA was denatured with 0.4 N NaOlnd 0.6 M NaC1 for 30 min, then washed oncewith 1.5 M NaC1 and 0.5 M Tris-HC1 for 30 min. DNA was transferred to Gene Screen Plus membrane (NEN-DuPont) by capillary blot, again denatured witb.4 N NaOH for 1 min, and then neutralized with 0.2 M Tris-HC1 (pH 7.0). DNA on membrarwas prehybridized for 1 h at 370C in 6 x SSC, 5 x Denhardt's solution, 1% SDS, containing 100 pg/ml of denatured salmon sperm DNA 30 (Sigma). Oligonucleotide probes (oligcN or oligo AF; 10 ng) were 3 2 P-5-end-labeled with 20 units of T4 kinase and 40 pCi 32 P-(-ATP for 30 min at 37*C. Unincorporated nucleotides were removed by centrifugatbn of the reaction mix through a minispin column (Worthington Biochemical Corp., Freehold, NJ). Hybridization was performed overnight at 370C. Membranesvere washed twice for 5 min each tine in 2 x SSC at room temperature, twice fol30 min in 2 x SSC, 0.1% SDS at 45'C, and once in 0.1 x SSC 58 WO 99/60108 PCT/US99/10731 for 30 min at room temperature. After washing, hybrids on membranes were analyzed autoradiographically by exposure to x-ray film. Analysis of 3CFTE29o- DNA shows replacement of the endogenous mutar(F508) sequences with the exogenous normal fragment as evidenced by PCR amplification of genomic DNA and allele-specific 5 Southern blot hybridization. PCR primers, one inside (CF1), and one outside (CF6) the region of homology (491 bp), were used to test whether the amplified DNA band was possibly due to amplification of any residual DNA fragment remaining in the cell after the transfection or by possible random DNA integration. A 687 bp fragmentcontains normal CFTR sequences while the 684 bp fragment is generate from AF508 CFTR DNA. To determine whether endogenous AF508 sequences were replaced with .0 exogenous normal CFTR sequences, we analyzd aliquots of the 687 or 684 bp amplification fragments by Southern hybridization using 3 2 P-end-labeled DNA probes specific for the AF508 or wild-type sequences (Table 1). h addition, the 687 bp fragment was PCR amplified by using the CF6 primer and a primer specific for either AF508 (oligo AF) or normal sequences (oligo N). The second round of DNA amplification with the CF1/oligo N or AF primer pair combination yields 300/299 bp fragments, 15 respectively. With the CF1/oligo N primer pair combination, a fragment will be dettEd only if the mutant DNA has been replaced by normal sequences. Fulter confirmation of homologous DNA recombination was tested by allele-specific Southern blot hybridization of the 300/299 bp fragments. Analysis of cytoplasmic RNA to detect normal exon 10 sequences in CFTR mRNA, verify that the homologous DNA recombination was legitimate and that normal CFTR mRNA is expressed in the 20 cytoplasm. To test whether the PCR geerated DNA fragments were exclusively CFTR mRNA-derived, primers in exon 9 (CF1 7) and allele-specific (normal, oligo N o0F508, oligo AF) primers in exon 10. Ths amplification with priners CF17/N yields a 322 bp normal fragment only if transcription of homologously recombined DNA has occurred. A 321 bp DNA fragment would be generated if thAF508 mutation were present. Furthermore, Southern hybridization analysis with allele-specific 32 P-end-labeled probes 25 differentiald between normal and AF508 mutant sequences and were also used to confirm expression of wild-type CFTR mRNA in the cytoplasm. Homologous recombination between the targeting polynucleotide coprising WT CFTR DNA and AF508 mutant cellular DNA allelic targets was evaluated by analysis or cellular DNA and RNA isolated from transfected and nontransfected 3CFTE29o-cell cultures. Nuclear genomic DNA and cytoplasmic RNA 30 were isolated 6 days after transfection, CFTR exon I sequences were amplifiaby PCR. Oligonucleotide primers (Table 1) were used to amplify the region of CFTR DNA spanning exon 10. One PCR primer (C I) was within the region of homology defined by the 491 bp DNA fragment (sense primer), and the other (CF 6) was outside the homologous region in the 3' intron (antisense primer). This DNA amplification reaction produces a 687 bp fragment with normal human CFTR DNA or a 684 bp fragment if the DNA 59 WO 99/60108 PCT/US99/10731 contains the AF508 mutation, as shown in Fig. 7A. Southern hybridization wasarried out on the 687/68 bp DNA fragments generated from amplification of genomic DNA from cell cultures by microinjection or by transfection with the protein-DNA-lipid complex, shown in Fig. 7B. A probe consisting of "P-end labeled oligonucleotide DNA that hybridized only to DNA sequences generated from a normal exon 1 0 5 was used. DNA from all microinjected and transfected cells produced specific hybrids as evidenced by autoradiographic hybridization. For cells microinjected with the 491 nucleotide fragmerifio. 7B, lane 2), the present of normal exon 10 sequences indicated homologous replacement at least a frequency of $ 2.5 x 10'. This result indicates at least one correctly targeted homologous DNA replacement in about 4000 microinjected nuclei. Other similar experiments using either electroporation or protein-DNA-lipid 10 transfection to transfer tle recA-coated 491 nucleotide CFTR DNA fragments also showed homologous recombination with the normal CFTR sequence in transfected CF cells. No hybridization was observed in control nontransfected (or mock-injected 3CFTE29o- cells). In each cell transfectedith normal CFTR DNA, analysis of the genomic DNA in a second round of allele-specific amplification of the 681/684 bp fragments with primers CFI/oligo N (Table 1) clearly showed the 300 bp fragment expectevIhen wild-type 15 CFTR sequences are present, as shown inFig. 8A. Fragments were detected for control 16HBE14o- cell (Fig. 8A, lane 2) and cells transfected with recA-coated DNA (Fig. 8A, lanes 5 anc.6 A 299 bp fragment (AF508-specific primer ends one base closer to the CF1 than the oligo N) was detected in DNA from nontransfected 3CFTE29o- cells amplified with CF1/oligdF primers (Fig. 8A, lane 4). No fragment was detected in DNA from nontransfected 3CFTE29o- cells reamplified with the lc/oligo N primers (Fig. 8A, 20 lane 3). Allele-specific Southern blot hybridization of these fragments with the 32 P-end-labeled oligo N probe resulted in autoradiographic hybridization signals from control normal and transfected CF cells (Fig 8B, lanes 1, 4, and 5) but not from DNA of nontransfected CF cells amplified tbi CF1 and oligo-N or -AF (Fig. 8B lanes 2 and 3). We tested whether any residual 491 nucleotide DNA fragments, which might remain in the cell after 6 days could act as a primer for the PCFeaction, genomic 3CFTE29o- DNA was 25 incubated with an equivalent number of recA-coated DNtfragments (10-104) introduced by microinjectim (Fig. 9). One antisense primer contains the wild-type normal (N) sequence while the other contains the AF508 (AF) mutation. Amplification wih the CFI/AF primer combination gives a 299 bp fragments when the AF508 mutation is present. No DNA fragment product was detected when the CF1/N primer combination we tsed with control nontransfected 3CFTE29o- DNA (Fig. 9, lane 2). However, when the 30 CF1/AF primer combination was used for DNA amplification in nontransfected 3CFTE29o- cells, a DNA product of the expected size (299 bp) was produced (Fig. 9, lane 1). These results indicate that all residual 491 nucleotide DNA fragments which might remain in the cells after 6 days of culture were incapable of competing with the CF1 PCR primers in the PCR anfication of the 687/684 bp fragments. Table 1 35 PCR Primers and Oligonucleotides 60 WO 99/60108 PCTIUS99/10731 Oligonuclectide DNA Strand DNA Sequence CF1 S 5'-GCAGAGTACCTGAAACAGGA CF5 A 5'-CATTCACAGTAGCTTACCCA CF6 A 5'-CCACATATCACTATATGCATGC 5 PCR Primers and Oligonucleotides Oliqonuclectide DNA Strand DNA Sequence CF17 S 5'-GAGGGATTTGGGGAATTATTTG OLITGO N A 5'-CACCAAAGATGATATTTTC OLIGO aF A 5'-AACACCAAGATATTTTCTT 10 Notes: (1) CF1 and CF5 PCR primers were used to synthesize the 491 b.p fragment used for thetargeting polynucleotide. (2) CF1 and CF6 PCR primers were used to amplify the 687/684 bp CFTR fragment. (3) The CF17 primer is located at the 5' end of exon 9 and was used for amplification of first 15 strand cDNA derived from CFTR mRNA. (4) Oligo N and Oligo AF are allele-specific probes and can also be used as allele-specific PCR primers for amplifying the 300/299 bp fragments (DNA analysis) and the 322/321 bp fragments (RNA analysis). (5) Sense (S) and antisense (A) primers are designated under DNA Strand and indicate the 20 sense of the strand relative to the transcribed direction (i.e., the CFTR mRNA). The corrected CFTR DNA must also be expressed at the mRNA level for normal function to be restored. Therefore, cytoplasmic CFTR mRNA was analyzed for the presence of a normal CFTR RNA sequence in the AF508 region of exon 10. Cytoplasmic RNA was isolated from the cells, reverse transcribed with DNA polymerase and PCR-amplified as first-strand cDNA. This amplification was 25 performed with a PCR primer located in exon 9 (CF17, sense) and CFTR allele-specific PCR primer in exon 10 (oligo N or AF, antisense). The exon 10 primer contains the CF mutation site, and the resulting fragment is 322 bp in normal DNA or 321 bp in DNA containing the AF508 mutation. Amplification of genomic DNA is eliminated by using primers that require amplification across intron/exon boundaries. Amplified cDNA generated from normal control 16HBE140- cells and 30 experimentally transfected cells yielded DNA product fragments with the CF17/oligo N, whereas nontransfected 3CFTE29o- cells only showed a DNA fragment after amplification with the CF17/oligo AF primers but not with the CF17/oligo N primers. Cells electroporated with wild-type 491-mer CFTR DNA showed the presence of wild-type CFTR mRNA. In addition, protein-DNA-lipid-transfected 3CFTE29o- cell cultures also showed the presence of wild-type CFTR mRNA in cells transfected with 61 WO 99/60108 PCT/US99/10731 the recA-coated 491 nucleotide fragment. Southern hybridization of the 322/321 bp cDNA fragments with the 1 2 P-end-labeled N oligonucleotide DNA probe showed the specificity of the PCR amplification and produced specific autoradiographic hybridization signals from all cell cultures transfected with recA-coated 491 nucleotide targeting polynucleotide. No autoradiographic hybridization signals were 5 detected in nontransfected 3CFTE29o- cells amplified with the CF17/oligo N or oligo AF primers. These analyses verify that the genomic DNA homologously recombined with the WT 491-mer DNA at the AF508 CFTR DNA locus resulting in RNA expressed and transported to the cytoplasm as wild type CFTR mRNA. This evidence demonstrates that human CF AF508 epithelial cells CFTR DNA can homologously 10 recombine with targeting polynucleotides comprising small fragments of WT CFTR DNA resulting in a corrected genomic CFTR allele, and that a recA-coated targeting polynucleotide can be used in transfection reactions in cultured human cells, and that cystic fibrosis AF508 mutations can be corrected in genome DNA resulting in the production of normal CFTR cytoplasmic mRNA. Taken together, the data provided indicates that 491-mer ssDNA fragments can find their genomic 15 homologues when coated with recA protein and efficiently produce homologously targeted intact cells having a corrected gene sequence. Analysis of CFTR in cytoplasmic RNA and genomic DNA by allele-specific polymerase chain reaction (PCR) amplification and Southern hybridization indicated wild-type CFTR DNA sequences were introduced at the appropriate nuclear genomic DNA locus and was expressed as CFTR mRNA in transfected cell cultures. Thus, in human CF airway epithelial cells, 20 491 nucleotide cytoplasmic DNA fragments can target and replace the homologous region of CFTR DNA containing a 3 bp AF508 deletion. Correctly targeted homologous recombination was detected in one out of one microinjection experiment with recA-coated targeting polynucleotide, two of two different electroporation experiments with recA-coated targeting polynucleotide, and one of one lipid-DNA-protein complex transfection 25 experiment with recA-coated targeting polynucleotide. Taken together, these 4 separate experiments strongly indicate that homologous recombination with recA-coated targeting polynucleotides (491-mer CFTR DNA) is feasible for treatment of human genetic diseases, and can be performed successfully by using various methods for delivering the targeting polynucleotide-recombinase complex. EXAMPLE 4 30 Homologous recombination in procaryotic cells In order to study the biological consequences of the cssDNA probe:target hybrid DNA structures in cells, we developed a simple and elegant assay to rapidly screen for in vivo homologous 62 WO 99/60108 PCT/US99/10731 recombination events in Escherichia coli. The principle of this assay is to screen for the recombinogenocity of hybrid structures formed between a dsDNA plasmid target carrying a 59 bp deletion in the lacZ gene (pRD.59) and cssDNA probes from the wild type lacZ (IP290) gene by introducing these pre-formed protein-free hybrids into E. coli by electroporation (Figure 10). 5 Homologous recombination frequencies are scored by plating transformed cultures in the presence of a chromogenic substrate (X-gal) so that recombinant bacterial cells (carrying plasmids that encode a wild type lacZ gene resulting from homologous recombination) appear blue. DNA plasmids and DNA probes: The plasmid pRD.59 was made from the 2.9 kb cloning vector pBluescript IISK(-) (pRD.0) (Stratagene). The pRD.0 DNA was linearized at a unique EcoRi site in the 10 polylinker region of the lacZ gene and digested with mung bean nuclease (Boehringer-Mannheim). The plasmids were then ligated and transformed into the RecA(-) E. coli host XL1-Blue (Stratagene). The resulting alpha peptide mutant clones were screened for lack of alpha-complementation of B galactosidase activity, which results in white colonies when grown on plates containing X-gal and IPTG (Sambrook et al., 1989). Plasmid DNAs recovered from white colonies by a mini-prep procedure 15 (Qiagen) lacked the unique EcoRI site, as well as the Xhol and Xbal sites. These mutant clones were then sequenced using Sanger dideoxy sequencing methods (Sequenase Kit version 2, USB) to determine the length of the deletion. Several clones containing deletions ranging from 4 bp to 967 bp were sequenced and named pRD for plasmids with an EcoRI deletion. The cloning vector pBluescript IISK(-) was named pRD.0 because it does not contain any deletions. 20 All samples of the plasmid DNA were then prepared by the Qiagen Maxi-Prep (Qiagen) procedure from strain of XL1-Blue (Stratagene) containing the plasmids. The cultures were grown on Luria-Broth (LB) media (Sambrook, et al., 1989) containing 100 pg/ml ampicillin. Recovered plasmids were more than 90% negatively supercoiled Form I DNA as judged by agarose gel electrophoresis. Biotinylated cssDNA probes were made from a fragment of the normal pBluescript IISK(-) plasmid. 25 The plasmid DNA was linearized with BglI and run on a 1% agarose gel in 1X TAE. After ethidium bromide staining, the 1.6 kB fragment band was excised from the gel and purified using the Qiaex 11 gel purification method (Qiagen). This 1.6 kb fragment was diluted 1:20 and then used as a template for PCR. The PCR reaction mixture contained biotin-14-dATP (GIBCO-BRL) in order to synthesize IP290, a 290 bp biotinylated cssDNA probe homologous to the LacZ region of pRD.O. In addition, 30 pRD.59 was linearized with Bgll and the 1.55 kb fragment was purified in the same manner as the pRD.0 1.6 kb fragment. Using the same primers that were used to synthesize IP290, the pRD.59 1.55 kb fragment was used as a template for PCR to synthesize DP231, a 231 bp biotinylated cssDNA probe homologous to the LacZ region of pRD.59. It is missing the 59 base pair sequence that flanks the EcoRI site. Biotinylated cssDNA probe CP443 was made in the same manner except that pRD.0 63 WO 99/60108 PCT/US99/10731 was linearized with Dral and different primers were used. CP443 is completely homologous to pRD.0 and pRD.59 in a region outside of the LacZ gene RecA mediated cssDNA targeting reactions and purification of probe:target DNA hybrids: Before targeting, biotinylated cssDNA probes (70 ng) were denatured by heat at 980C for 10 minutes, cooled 5 immediately in an ice-water bath, and then centrifuged at 40C for 10 seconds to recover all liquids. Reactions without cssDNA probe contained equivalent volumes of water. The denatured cssDNA probes were then coated with RecA protein (Boehringer-Mannheim) in Tris-acetate reaction buffer (Cheng et al., 1988; 10 mM Tris-acetate (pH 7.5), 1 mM dithiothreitol, 50 mM sodium acetate, 2 mM magnesium acetate, 5% (v/v) glycerol) with 2.43 mM ATPS for 15 minutes at 37 0 C in a 10 pl volume. 10 Reactions without the RecA protein contained equivalent volumes of RecA storage buffer (20 mM Tris HCl, pH 7.5, 0.1 mM EDTA, 1 mM DTT, and 20% glycerol). The RecA mediated targeting reactions were performed by adding 1- 4 pg of the appropriate plasmid DNA in an aqueous solution containing 22 mM magnesium acetate, bringing the final magnesium concentration to 11 mM and the final reaction volume to 20 pl. The reaction was incubated for another 15 60 minutes at 370C. At the end of the targeting reaction, SDS was added to a final concentration of 1.2% to deproteinize the complexes. If further enzymatic treatments were necessary on the targeted complexes, 3 volumes of phenol:choloform:isoamyl alcohol (Sigma), shaken on a Multi-Tube Vortexer (VWR) for 4 minutes at 4*C, and centrifuged for 5 minutes at 40C. The supernatant was recovered, placed in a new tube, and 20 extracted with 1 volume of chloroform. The mixture was shaken for 2 minutes at 4*C, and centrifuged for 5 minutes at 4*C. The supernatant was recovered, containing the purified targeted complexes. Detection of probe:target DNA hybrids: After deproteinization, the complexes were run for 20 hours at 30 V on a 20 cm by 25 cm 1% agarose TAE gel (GIBCO-BRL) at room temperature. The gels were visualized by staining in 1 pg/ml ethidium bromide and then cut down to 11 cm by 14 cm before they 25 were soaked in 1OX SSC and transferred to positively charged Tropilon membranes (Tropix) by Southern blotting method under non-denaturing conditions. Blots were then UV cross-linked (Stratalinker). Biotinylated cssDNA probes and probe:target hybrids were detected using the Southern-Light System (Tropix). The nylon bound DNA blots were treated with avidin conjugated to alkaline phosphatase, 30 followed by the chemiluminescent substrate, CDP-Star (Tropix), in conditions described by the manufacturer. Blots were exposed to X-ray film (Kodak) for varying times (1 minute to 8 minutes) and developed. 64 WO 99/60108 PCTIUS99/10731 Electroporation of probe:target DNA hybrids into metabolically active E. coli cells: After purification of targeted complexes, 40 pl of electro-competent RecA(+) and/or RecA(-) E. coli (Dower et al., 1988) was added to 30-200 ng of the targeted complexes in a chilled microfuge tube. The RecA(+) cells were BB4 (Stratagene) and the RecA(-) cells were XL1-Blue (Stratagene). The mixture was incubated 5 on ice for 1 minute. This mixture was then transferred to a chilled 0.1 cm gap electroporation cuvette (Bio-Rad) and electroporated under the following conditions: 1.3 V, 200 ohms, 25 pF on a Bio-Rad Gene Pulser. The time constant ranged from 4.5 - 4.7 msec. Immediately afterwards, 1 mL of SOC media (Sambrook, et al., 1989) was added and the mixture was transferred into a 10 mL culture tube. After all the electroporation groups were finished, the tubes were shaken at 225 rpm at 37 0 C for 1 10 hour. Appropriate amounts were plated onto LB agar plates which already contained 100 pg/ml ampicillin (Sigma), 20 pg/mI X-gal (GIBCO-BRL), and 48 pg/ml IPTG (GIBCO-BRL), and incubated at 37*C overnight. Screening for homologous DNA recombination in LacZ: After overnight incubation (approximately 16 hrs.), colonies were counted to determine electroporation efficiency and scored for any blue colonies 15 in plates. Blue colonies were scored if they resembled blue colonies displayed by the control plasmid pBluescript Il SK(-), which is able to undergo alpha-complementation and produce blue colonies. Blue colonies were serially propagated on AIX plates at least twice to confirm recombinant stability as monitored by consistency of color. When the colonial streaks displayed a homogeneous color, plasmids were isolated by a mini-prep and digested with EcoRI, Xhol, and Pvull to confirm 20 homologous recombination of the plasmid at the DNA level. EcoRI and Xhol sites are restored if homologous recombination has occurred. Pvull restriction sites which flank the LacZ region contains the 59 base pair deletion; if recombination has occurred, this fragment will be significantly larger than fragments lacking the 59 base pairs after digestion with Pvull. RecA mediated cssDNA targeting to negatively supercoiled dsDNA substrates containing deletions: 25 Stable probe:target hybrids formed in the RecA mediated targeting reaction between the biotinylated RecA coated cssDNA probes IP290 and the negatively supercoiled Form I dsDNA targets pRD.59, which contain a 59 base pair deletion respective to the cssDNA probe, were monitored by chemiluminescent detection of biotinylated hybrids (Figure 11). The presence of a sizable region of non-homologous nucleotide sequences (59 bp) in the cssDNA probe IP290 does not significantly 30 affect the ability of the RecA coated cssDNA probe IP290 to form stable probe:target hybrids with pRD.59 in comparison to completely homologous dsDNA pRD.0 (Figure 11, lane 3 and 6). In each reaction, under these conditions, the presence of the RecA protein was absolutely required for hybrid detection (Figure 11, lane 2 and 5). 65 WO 99/60108 PCT/US99/10731 Probe:target DNA hybrids formed when the RecA coated biotinylated cssDNA probe IP290 is hybridized to the completely homologous dsDNA target pRD.0 differ from probe:target hybrids formed when the same cssDNA probe is hybridized to the dsDNA target pRD.59 containing a 59 base pair deletion with respect to IP290. While more than 90% of both the dsDNA targets exist as negatively 5 supercoiled Form I DNA, when hybrids formed between pRD.0 and RecA coated cssDNA probe IP290 are deproteinized, the probe:target hybrids migrate to a position that is similar to the migration of Form II, relaxed circular dsDNA, in 1% agarose gel in 1X TAE buffer (Figure 11, lane 3 and 6), and there was no evidence of probe:target hybrids that co-migrate to Form I DNA on a 1% agarose gel (Figure 11, lane 3). This probe:target hybrid is referred to as a relaxed Form I* hybrid or a rl* hybrid because 10 the hybrid has the same elelctrophoretic mobility as relaxed circular DNA. In contrast, when the RecA coated cssDNA probe IP290 was hybridized to the dsDNA target pRD.59, which as a 59 bp deletion with respect to the probe, two different probe:target hybrids were apparent. One has an electrophoretic mobility comparable to that of Form I supercoiled dsDNA (Figure 11, lane 6) while the other migrates to the same position as the rI* hybrid. These two forms appear to be present in equal 15 amounts as indicated by the signal from chemiluminescent DNA detection. This probe:target hybrid is referred to as a Form I* hybrid or 1* hybrid, differentiating it from Form I DNA because it is targeted with RecA coated cssDNA probe. In order to exclude the possibility that it is the structure of the dsDNA target that creates the formation of two major probe:target hybrid products, the cssDNA probe DP231 was hybridized to pRD.59. The cssDNA probe DP231 is completely homologous to the mutant 20 region of the LacZ gene in pRD.59. The only probe:target hybrid detected has the electrophoretic mobility of Form I dsDNA, the rI* hybrid (Figure 11, lane 8). In addition, when the cssDNA probe CP443, which is completely homologous to a region outside of the 59 base pair deletion, was hybridized to pRD.59, only the rI* hybrid product was detected (Figure 11, lane 10). Thus, when the RecA coated cssDNA probes are targeted to homologous sequences, only the rl* hybrid is present, 25 but when it is targeted to homologous sequences with relatively short heterologies, two forms of hybrids, rI* and I* hybrids are formed in apparently equivalent amounts. Recombinogenicity of probe:target DNA hybrids: To study the biological consequences of the probe:target hybrid structures , we assayed for putative homologous recombination events in E. coli by the electroporation assay (described in Figure 10). 30 Figure 12 shows the percentage of potential recombinant blue colonies formed when IP290 probe:pRD.59 target hybrids were electroporated into RecA+ and RecA- cells. Blue colonies only arose when deproteinized hybrids formed with pRD.59 and cssDNA probe IP290 are introduced into RecA+ E. coli cells. Control experiments performed with cssDNA probes homologous to the mutant LacZ region of pRD.59 (DP231) and homologous to a region outside of the LacZ gene (CP443) did not 35 yield any blue colonies. (Figure 12). In addition, when all of these hybrids were transformed into 66 WO 99/60108 PCT/US99/10731 RecA(-) hosts, no blue colonies were produced from any type of hybrid, indicating the the recombinogenic effect is also dependent on endogenous RecA protein produced in the cell. Thus only the cssDNA probe containing the 59 base pair correction produces recombinogenic clones in bacterial host cells that are RecA(+). 5 When potential homologous recombinant blue colonies were propagated by streaking out on AIX plates, only 50% of the colonies were blue. When a blue colony from the first streak was propagated by recombinant streaking, the colonies remained stably blue over several generations. If plasmid DNA was isolated from third generation propagations and then transformed into RecA(-) cells, this resulted in blue colonies which remained stably blue on continued propagation. Of the potential 10 recombinants that have been rigorously screened by restriction enzyme digestion, at least 67% of the plasmids recovered from blue colonies are true homologous recombinants. This was deterimined by the restoration of EcoRI and Xhol restriction sites, and a Pvull digest of the DNA shows a fragment that migrates at a higher molecular weight than fragments which are missing the 59 base pair region. This is consistent with the view that only one strand is exchanged in these hybrids to form 15 heteroduplex targets and that upon replication one strand will produce a plasmid that contains the 59 base pair correction while the other does produces the mutant pRD59 plasmid. As outlined in Example 5, we show that the recombinogenicity with probe:target hybrids of cssDNA probes and dsDNA targets containing deletions is associated with the re-annealing of regions of cssDNA probe that can not hybridize to dsDNA targets, by creating internal homology clamps (Figure 20 13). EXAMPLE 5 Enhanced homologous recombination with targets containing insertions and deletions containing internal homology clamps An in vitro DNA hybridization reaction that allows the pairing of RecA-coated complementary single 25 stranded (css) DNA probes to homologous regions in linear duplex target DNA has been used to study the effects of heterologies within the regions of homology between the probes and target DNA. In cssDNA targeting reactions catalysed by RecA protein, cssDNA probes are kinetically trapped within the duplex DNA target at homologous sites and form a highly stable four-stranded DNA hybrid structure. After removal of RecA protein, this homologous recombination reaction can be trapped at 30 the DNA pairing step. The effect of defined heterologous insertions or deletions in linear duplex targets on the pairing of RecA-coated cssDNA probes was determined for heterologies ranging from 4 to 967 bp. We demonstrate that small deletions and insertions up to 10% of the total cssDNA probe 67 WO 99/60108 PCTIUS99/10731 lengths, ranging from 215 -1246 bp do not significantly affect DNA pairing. Furthermore both insertions and deletions of the same size in the cssDNA probe have the same effect on DNA pairing. Moreover, large deletions, up to 967 bp, can be tolerated in deproteinized hybrids form with a RecA coated 1.2 kb cssDNA probe. The stability of these hybrids with heterologous sequences within the 5 homologous paired region is due to the re-annealing of the cssDNA probes to each other within the DNA hybrid producing a novel four-stranded heteroduplex DNA intermediate that contains a novel internal base-paired homology clamp. Preparation of ds target substrates: A series of plasmid DNA targets with defined deletions were constructed by linearization of the plasmid vector pBluescript IISK(-) (Stratagene) at a unique EcoRI 10 restriction site in the polylinker region following digestion with mung bean exonuclease (Boehringer Mannheim), DNA ligation, and subsequent transformation into XL1-Blue E coli (Stratagene) by standard methods. The resulting clones were sequenced using Sanger dideoxy sequencing methods (Sequenase Kit version 2, USB) to determine the extent of deletion. A series of plasmids with deletions ranging from 4 to 967 bp were prepared and named for the extent of size of the deletion 15 (see Figure 15). The size of the parent plasmid, pBluescript IISK(-), referred to as pRD.0 in this study, is 2960 bp. Plasmid DNA was prepared by a modified alkaline lysis procedure with anion-exchange purification (Qiagen). The DNA was further purified by phenol-chloroform-isoamyl alcohol extraction (24:25:1) (SIGMA) and ethanol precipitation, and then resuspended in TE (10 mM Tris HCI, pH7.5, 1 mM EDTA).buffer. These preparations contained greater than 90% Form I DNA. Preparations of 20 linearized Form Ill DNA were made by digestion of the plasmids at a unique Scal restriction site outside the polylinker, followed by phenol-chloroform-isoamyl alcohol extraction (SIGMA), chloroform extraction, ethanol precipitation, and resuspension in TE buffer. Preparation of cssDNA probes: Biotin-labeled probes homologous to pRD.0 were synthesized by PCR with incorporation of biotin-14-dATP using previously described methods where the molar ratio of 25 unlabelled dATP to biotin-labelled dATP was 3:1 (Griffin & Griffin, 1995). Primer pairs flanking the polylinker region of pRD.0 or analogous plasmids with a deletion were chosen to produce PCR fragments which span the deletion in the target plasmids. In addition a control PCR fragment (CP443) primer pair flanking sequences outside the polylinker was selected for production of a probe homologous to all clones in the plasmid series. The oligonucleotide products were purified by 30 membrane ultrafiltration using Microcon 100 filters (Amicon). Targeting of cssDNA probes to dsDNA targets in solution: cssDNA targeting was performed essentially as described in Sena & Zarling (1993), with the exception that cssDNA probes were synthesized and labeled by PCR in the presence of biotin-14-dATP (GIBCO/BRL), as indicated above. In each reaction 70 ng of biotin-labelled RecA-coated cssDNA probe was reacted with 1 pg of Scal 68 WO 99/60108 PCT/US99/10731 digested target DNA, resulting in cssDNA probe:target ratios of 1:1 (for 215 bp cssDNA probes) to 1:5 (for 1246 bp cssDNA probes). The products of the targeting reactions were deproteinized by treatment with SDS (1.2% final concentration) or phenol:chloroform: isoamyl alcohol (24:25:1) and chloroform extraction and then separated by electrophoresis on 1% agarose gels in TAE buffer. The 5 gels were run at 2V/cm at room temperature in the absence of ethidium bromide for 20 hours. After electrophoresis, gels were stained in 1 pg/mi ethidium bromide for 15 min. The DNA was transferred under non-denaturing conditions (10X SSC) onto nylon membranes (Tropix) and cross-linked using a Stratalinker (Stratagene) on the auto-crosslink setting. The extents of biotinylated cssDNAprobe:target hybrid formation was measured by quantitating the amount of biotin-labeled 10 probe DNA that co-migrates with dsDNA target DNA following electrophoretic separation of these biotinylated probe:target hybrid products from free unhybridized probe DNA. The amount of biotinylated probe DNA in probe:target complexes was visualized with a chemiluminescent substrate conjugated to streptavidin (CDP-STAR) (Tropix) after exposure to XAR-5 film (Kodak). The levels of exposure were analyzed by densitometry and quantitated using the software package, NIH Image. 15 In each case the relative level of hybrid formation with heterologous targets was expressed as a percentage of the level of hybrid formation of standardized reactions with a completely homologous probe and target. These values were normalized to the level of hybrid formation that occured with control probe CP443 which hybridizes to all of the plasmid targets in a region away from the heterology. The data generally represent averages of at least three separate measurements from 20 three independent targeting reactions. Nomenclature and Assay for RecA-mediated pairinq of cssDNA probes to dsDNA targets.: To investigate the effects of heterologous insertions and deletions on homologous pairing of cssDNA probes to double-stranded linear plasmid DNA, we employed a modification of an in vitro DNA targeting assay described in Sena and Zarling (1993). The target DNAs used in this study are a series 25 of plasmid DNA constructs that contain defined deletions at the unique EcoRI site in pRD.0 (pbluescriptllSK(+), Stratagene Figure 14A). Plasmid targets (pRD.4 - pRD.967) are named for the size of deletion in bp at the EcoR1 site. CssDNA probes were made and labelled with biotin-14-dATP by PCR using primers which symetrically flank the deleted region of plasmids in the pRD series. CssDNA probes made from pRD.0 that were targeted to plasmids containing deletions are called 30 insertion probes and named for the length of the probe in bp. For example, IP290 is a 290 bp cssDNA probe that contains an insertion with respect to a target containing a deletion, but is completely homologous to pRD.O. A cssDNA probe made from pRD.59 and targeted to pRD.0 is called DP231, since it contains a deletion with respect to pRD.0, but is completely homologous to pRD.59. 69 WO 99/60108 PCT/US99/10731 After the hybridization of RecA-coated cssDNA probes with dsDNA targets, the reactions products were separated by agarose gel electrophoresis. The extent of formation of stable deproteinized cssDNA probe:target hybrid was measured by the quantitation of the amount of biotinylated cssDNA probes that co-migrated with the dsDNA targets. In each case the level of probe:target formation 5 between a totally homologous probe and target was normalized to 100%. Previous studies have shown that efficient cssDNA targeting is completely dependent on RecA protein, the nucleotide co factor, specific to homologous DNA targets and that formation of deproteinized stable probe:target hybrids also requires both cssDNA strands (Sena and Zarling, 1993, Revet et al, 1993). Furthermore we targeted Scal-digested pRD.0 with two synthetic RecA-coated 121-mer cssDNA oligonucleotides 10 homologous to the region symetrically spanning the EcoR1 site in pRD.0 and demonstrated that both cssDNA strands are required for stable hybrid formation with linearized pRD.0 targets (data not shown). Stable cssDNA probe:target hybrids are formed in linear dsDNA targets with deletions at internal sites. To determine if a target DNA deletion affects the reaction kinetics of RecA-mediated cssDNA pairing 15 to linear DNA targets, we measured the relative amount of deproteinized cssDNA probe:target hybrid formation over time in reactions using cssDNA probe IP290 with either a completely homologous linear target, pRD.0 or a target carrying a 59 bp deletion, pRD.59. Probe IP290 symetrically spans the 59 bp deletion in pRD.59. Figure 15B shows that in steady state hybrid reactions, the maximum level of stable hybrid formation when RecA-coated IP290 is targeted to pRD.59 is 62% of the steady state 20 level obtained with the fully homologous target pRD.O. Furthermore steady state levels of hybrid formation occurs within 45 minutes with fully homologous pRD.0 targets, but requires 2 hours for pRD.59 targets. Thus, in all subsequent experiments RecA-coated probes were hybridized for 2 hours at 370C with the linear target DNAs. The effect of duplex DNA target deletions on the formation of deproteinized cssDNA probe: target 25 hybrids was determined by hybridizing RecA coated cssDNA probes which span the deleted regions in pRD.4 - pRD.298 on DNA targets linearized by Scal (Figure 15A). The relative amount of biotinylated probe:target hybrids formed with each of these targets was compared with the amount of cssDNA probe target hybrids formed with pRD.O. These values were normalized to the level of hybrid formation obtained with the control probe, CP443, which is homologous to a region away from the 30 deleted regions or pRD.0 and thus, is completely homologous to all target DNA substrates used in this study. Our initial studies tested the effect of small target deletions on targeting efficiency using either cssDNA probes IP527 or IP407 (Figure 15B and 15C). Because the 5'- and 3-termini of both of these cssDNA probes are approximately symmetric with respect to the 4 to 59 bp deletions, the differences in the 70 WO 99/60108 PCTIUS99/10731 efficiency of hybrid formation are not due to the effects of the position of the deletion with respect to the probe in relation to probe ends. As expected, in experiments using either the IP527 or IP407 we observed a decrease in the level of hybrid formation with an increase deletion size. These data also show that relatively small deletions (<i 25 bp) in the target do not dramatically affect the overall 5 targeting efficiency of cssDNA probes to linear targets and that the deletions have relatively the same effect on the hybridization on either IP527 and IP407. However when the size of the deletion is increased to 59 bp (11% of the length of IP527), the relative targeting efficiency of probes IP527 and IP407 drops to 61% and 33%, respectively. Furthermore the amount of the difference between the targeting efficiency mediated by these probes continues to increase linearly as the size of the deletion 10 increases (Figure 15D). This indicates that when the size of the deletion is >10% of the length of the probe the efficiency of RecA-mediated DNA targeting is governed by the amount of homology between the cssDNA probe and target, while deletions <10% of the length of the probe are well tolerated for any length of cssDNA probe. Similar effects are observed with smaller cssDNA probes IP452, IP290 (data not shown) and IP215 (Figure 16). 15 Heterologous insertions and deletions are similarly tolerated in the hybridization of cssDNA probes to linear dsDNA targets. Other studies by Bianchi and Radding (Cell 35:511-520 (1983)) in which RecA coated circular ssDNA was hybridized to linear duplex targets demonstrated that heterologous inserts in the ssDNA were tolerated somewhat better than when the insert was in the dsDNA, presumably because the inserts in ssDNA could be folded out of the way. In contrast, Morel et al (J. Biol. Chem. 20 269:19830 (1994)) used somewhat similar substrates and demonstrated that RecA-mediated strand exchange could bypass heterologies with equal efficiency whether the insert was in the ssDNA or dsDNA. Since the formation of stable cssDNA:probe target hybrids with internal sequences in linear dsDNA requires two cssDNA probe strands, we compared the effects of insertions in the cssDNA probe with having the same sized insertion in the dsDNA to determine how these internal heterologies 25 maybe accommodated within a four strand containing double-D-loop DNA structure. In these studies we compared the effects of 4 to 59 bp insertions in either the dsDNA target or cssDNA probe (deletion in target) using cssDNA probes ranging in size from 156 bp to 215 bp. We used this smaller cssDNA probe to maximize the effects of the insertion or deletion of these sizes. We prepared cssDNA probe IP215 from pRD.0 using PCR and targeted pRD.0, pRD.4, pRD.25, and 30 pRD.59 to measure the effects of insertions in cssDNA probes (target DNA deletion). Then using the same PCR primer set, we prepared cssDNA probes from templates pRD.0, pRD.4, pRD.25, and pRD.59 and then targeted pRD.0 to measure the effects of deletions in cssDNA (target DNA insertion). Figure 16 shows that both deletions and insertions of the same size have exactly the same effect on RecA-mediated cssDNA targeting and are equally tolerated and stable. 71 WO 99/60108 PCT/US99/10731 Large deletions in linear DNA are tolerated in cssDNA probe:target hybrids with linear dsDNA. To further define the extents of heterology that can be tolerated during cssDNA hybridization, we studied the effect of very large deletions, up to 448-967 bp on the targeting efficiency using a 1246 bp cssDNA probe (IP1246) (Figure 17A) . With target deletions in the range of 500 bp (approx. 50% of 5 the cssDNA probe length) there is only a slight reduction in the targeting efficiency achieved with this probe (80%), surprisingly the IP1246 can hybridize target DNA molecules bearing deletions up to 967 bp at a detectable efficiency (27%). When IP1246 is targeted to pRD.967, there are a total of 279 bp of homology between the cssDNA probe and target, with 147 bp 5' to the 967 bp insert and 132 bp 3' to the insert (Figure 17B). In order to account for such a high level of targeting efficiency with such a 10 large deletion, we predict that the 967 bp insert in the two in the cssDNA probe strands, which are homologous to each other, may interact with each other to stabilize this hybrid. Furthermore when using a large cssDNA probes of 1246 bp we can observe a visible shift the migration of the cssDNA probe:target hybrid in comparison to the linear dsDNA target. The positions of the migration of the of the 3.0 kb Scal -digested ds DNA marker are shown in Figure 17A. Note the 15 cssDNA probe:target hybrids formed with IP1248 migrate slower than each of the Scal-digested targets, but that cssDNA probe:target hybrids formed with CP443, a smaller probe migrate closer the positions of the formIll markers. The presence of this labelled slower-migrating species provides further evidence for the existence of the multi-stranded DNA hybrids. EcoR1 Restriction endonucleases cut duplex DNA in either homologous or heterologous cssDNA 20 probe:target hybrids. To further characterize cssDNA probe:target hybrids formed with heterologous DNA targets, circular plasmids pRD.0 and pRD.59 were hybridized with biotin-labelled probe IP290 and then deproteinized and digested with EcoRI. While plasmid pRD.0 contains a unique EcoR1 site in the region of homology between IP290 and pRD.0, the EcoR1 site is deleted in pRD.59 (Figure 14A). Digestion of cssDNA probe:target hybrids with EcoR1 indicates the restoration of Watson-Crick 25 pairing to form a fully duplex EcoR1 recognition site. Figure 18 shows both the ethidium bromide stained gel of the hybrid product of the targeting reaction (Figure 18A and 18B) and the corresponding autoradiograph that shows the electrophoretic migration of the biotin-labelled probes (Figure 18C and 18D). These data show that when RecA-coated IP290 is hybridized to the fully homologous pRD.0 plasmid all of the probe:target hybrids migrate to the position of fully relaxed DNA (Figure 18 A and C, 30 Lane 1). Furthermore, upon digestion with EcoR1 cssDNA:probe target hybrids can be completely cut, as shown in Figure 18 A and C, Lane 2. When similar reactions are performed with uncut pRD.59 targets, we found that not all of the probe:target hybrids are relaxed as with pRD.0 targets, as judged by the appearance of two bands corresponding to a pRD59 I* hybrid, where the hybrids co-migrate with Form I supercoiled DNA and a pRD59 rl* hybrid that migrates with relaxed targets (Figure 18B 35 and D, Lane 3). When these hybrids are digested with EcoRI we find that the pRD59 rl* hybrid is 72 WO 99/60108 PCTIUS99/10731 more susceptible to EcoRI cleavage than the pRD59 rI* hybrid (Figure 18B and D, Lane 4). This shows that there is a restoration of the EcoRl site in relaxed targets, but not in the non-relaxed I* hybrid. Since pRD59 targets do not contain an EcoRI site, cleavage by EcoRI can only be explained by re-annealing of cssDNA probe IP290 within the IP290 probe:target pRD59 hybrid. 5 To further characterize the structural differences between pRD59 rl* hybrids and pRD59 1* hybrids, cssDNA probe:target hybrids were formed between IP290 and pRD59, deproteinized and thermally melted for 5 mins at 370C, 450C, 55'C, and 650C, respectively. Figure 19 shows that pRD59 rl* hybrids are more thermostable than pRD59 1* hybrids. For both types of hybrids probe:target hybrids are completely dissociated after heating to 950C (data not shown). Taken together these data support 10 the structures of our models for hybrids (Figure 13). EXAMPLE 6 Homologous recombination targeting in fertilized mouse zygotes Ornithine transcarbamylase (OTC) is a mitochondrial matrix enzyme that catalyzes the synthesis of citrulline from ornithine and carbamylphosphate in the second step of the mammalian urea cycle. 15 OTC deficiency in humans is the most common and severe defect of the urea cycle disorders. OTC is an X-linked gene that is primarily expressed in the liver and to a lesser extent in the small intestine. Affected males develop hyperammonemia, acidosis, orotic aciduria, coma and death occurs in up to 75% of affected males, regardless of intervention. Two allelic mutations at the OTC locus are known in mice: spf and spf-ash, (sparse fur--abnormal skin and hair). In addition to hyperammonemia and 20 orotic aciduria, spf-ash mice can be readily identified by the abnormal skin and hair phenotype. The spf-ash mutation is a single-base substitution at the end of exon 4 that results in alternative intron exon splicing to produce an aberrant non-functional elongated pre-mRNA. Because of the clinical importance of OTC defects in humans, there is an intensive effort to develop in vivo methods to correct the enzymatic defect in the spf-ash mouse model. 25 We used the murine spf-ash model of OTC deficiency to test the ability of RecA-coated complementary single-stranded DNA (css) OTC probes to target and correct a single-base substitution mutation in fertilized mouse zygotes. A 230 bp RecA-coated cssDNA probe amplified from the normal mouse OTC gene was microinjected into embryos derived from matins of B6C3H homozygous spf-ash female with normal B6D2F1J males. After re-implantation of 75 embryos that were microinjected with 30 RecA-coated cssDNA into CD1 foster mothers, 25 developmentally normal pups (17 female and 8 male) were born. Sequence analysis of the genomic DNA isolated from tails of the male pups show that 3 out of 8 males were mosaic for a homologous recombination event at the spf-ash site in exon4 of the mouse OTC gene. Subsequent breeding of the three founder males with normal females 73 WO 99/60108 PCT/US99/10731 resulted in normal female F, progeny, thus demonstrataing germline transmission of the homologous recombinant allele as well as phenotypic correction in F, animals. These homologoous recombinant changes were stable in F 2 and subsequent generations. These studies illustrate cssDNA mediated high frequency homologous recombination in fertilized mouse zygotes to create subtle genetic 5 modifications at a desired target site in the chromosome. Preparation of RecA-coated probe: A 230 bp fragment from the normal mouse OTC gene was amplified by PCR with primers M9 and M8 from pTAOTC (Figure 20). The PCR fragment was purified on Microcon-100 columns (Amicon) and then extensively dialyzed. The M9-M8 amplicon was denatured by heating the fragments to 980C and then coated with RecA protein (Boehringer 10 Mannheim) at a ratio 3 nucleotides/ protein monomer. The final concentration of RecA-coated DNA in coating buffer (5 mM TrisOAc, pH 7.5, 0.5 mM DTT, 10 mM MgOAc, 1.22 mM ATP(S, 5.5 pM RecA) was 5 ng/ pL. RecA-coated filaments were made on the day of microinjection and then stored on ice until use. Transgenic Mice: Five superovulated B6C3H (spf-ash/spf-ash) 5-7 week old females (Jackson Labs) 15 were mated with five B6D2F1 males (Jackson Labs). Approximately 80-100 embryos were isolated from oviducts as described in Hogan et al. (1988). The female pronucleus of fertilized embryos was microinjected with 1-2 pl of RecA-coated M9-M8 cssDNA probe (5 ng/pL). Approximately 75 embryos survived the microinjection procedure and were then re-implanted into a total of three CD1 pseudopregnant foster mothers (Charles River). Pseudopregnant females were produced by mating 20 foster mothers with vasectomized CDI males (Charles River). DNA Analysis: Tail biopsies were taken from all founder mice after weaning at three weeks of age. Genomic DNA was isolated from tail biopsies using standard procedures. To obtain the sequence of the DNA at the OTC locus, genomic DNA was amplified with PCR using primers M10-M1 1 or M54 M1 1 that flank the cssDNA probe sequence to generate a 250 bp or 314 bp amplicon (Figure 20). 25 PCR fragments were sequenced manually using the Cyclist Exo- Kit (Stratagene), automatically on an Applied Biosystems Model 373A sequencer, or by a MALDI-TOF mass spectrometry system (GeneTrace Systems, Menlo Park, CA) Fertilized zygotes microiniected with RecA-coated DNA are viable. Plasmid pTAOTC1 carries a 250 bp segment of exon4 and surrounding intron sequences from the normal mouse OTC gene. A 230 bp 30 cssDNA probe OTC1 was prepared by PCR amplification of pTAOTC1 with primers M9 and M8. cssDNA probe OTC1 was denatured and coated with RecA protein as described herein. 74 WO 99/60108 PCT/US99/10731 Homozygous spf-ash/spf-ash female and hemizygous (spf-ash/y) males can be phenotypically identified by the appearance of sparse fur and wrinkled skin early in development. A cross between homozygous spf-ash/spf-ash B6C3H females and normal B6D2F1 males yields heterozygous phenotypically normal females and hemizygous males with sparse fur and wrinkled skin. The RecA 5 coated cssDNA OTC probe was microinjected into embryos made from the cross of B6C3H homozygous female spf-ash and normal males. The female pronucleus of approximately 80-90 fertilized zygotes was microinjected with 2 pl of a 5ng/pL solution of RecA-coated cssDNA probe OTC1. Of these, 75 embryos survived the microinjection procedure. To demonstrate that embryos that have been microinjected with RecA-coated cssDNA are viable, the embryos were re-implanted 10 into three pseudopregnant CD1 foster mothers. From this, 25 developmentally normal pups (17 female and 8 male) were produced. All of the female mice were phenotypically normal. The eight male mice (mouse # 7, 14,16,17,22,23,24, and 25) were all affected with sparse-fur and wrinkled skin to various degrees. RecA-coated cssDNA probe OTC1 recombines with the homologous chromosomal copy of the OTC 15 gene in fertilized mouse zygotes. To determine the genotypes of the 25 founder mice produced from microinjected embryos, genomic DNA was isolated from tail biopsies. Genomic DNA was amplified with either the primer set M10-M 11 or M54-M1 1 to produce either a 250 bp or 314 bp amplicon. By using these primer sets that flank the OTC1 probe, the DNA amplicon represents DNA from the endogenous OTC gene. PCR fragments from all of the eight mice and several female mice were 20 sequenced to determine the base sequence at the spf-ash locus to determine if a normal allele (G) or a mutant allele (A) was present in the genomic DNA. Figure 21 shows sequencing gels of representative reactions. The panel on the left side shows the sequence of the homozygous spf-ash females that donated the eggs to produce the fertilized zygotes where only the mutant base A is present at the spf-ash locus, as expected. The sequence of female mouse #8 that should be 25 heterozygous shows the presence of equal amounts of the bases G and A as expected. Male mice 7, 14 (shown), 23, 24,and 25 all showed only the mutant base A at the spf-ash locus, however male mice 16, 17, and 22 (shown) displayed both G (normal) and A (mutant) at the spf-ash locus. To eliminate the possibility of PCR artifacts during PCR cycle sequencing the base compositions of the samples was independently confirmed by mass spectrometry sequencing (GeneTrace, Menlo 30 Park). The relative (%) amounts of the A:G base composition at the spf-ash locus was also quantified and determined to be 70%:30% for samples from mouse #16 and #17 and 10%:90% for mouse #22. Since OTC is an X-linked gene the presence of mixed bases in male mice is likely the result of the mosaic animals produced of a mixture of mutant and gene corrected embryonic cells. 75 WO 99/60108 PCTIUS99/10731 Germline transmission of the gene corrected OTC allele. To determine if the gene corrected allele in the mosaic male founder mice (#'s 16, 17, and 22) could be passed through to the germline, these mice and a control hemizygous mutant male (#7) were bred with normal B6D2F1 females. In this cross, if the male donates a mutant spf-ash X chromosome, then the resulting female progeny will be 5 heterozygous spf-ash mutants. However if the male donates a normal (gene corrected) X chromosome the female progeny will be homozygous normal. In both cases the resulting F1 females will be phenotypically normal. The results of these crosses are summarized in Figure 22. In the control cross of hemizygous mutant male #7 with B6D2F1 females, all 14 female progeny were heterozygous, as expected. In test crosses of mosaic male mouse #17 and #22 with normal females, 10 all resulting female progeny (5 and 9, respectively) were heterozygous. However in the cross with mosaic male mouse #16, one out nine total female progeny was a homozygous normal female (mouse # 213), as determined mass spectrometry DNA sequencing (GeneTrace, Menlo Park), demonstrating the gene corrected allele in founder mouse #16 was tranmitted through the germline. To further verify that the F1 mouse #213 was, in fact, a germline-transmitted gene corrected 15 homozygous normal female, this mouse and a control heterozygous spf-ash/+ mouse were bred with normal males. In the control cross B with the heterozygous female, 50% of the resulting male F2 progeny should be mutant spf-ash/y hemizygotes that can be easily determined by the visualization of the sparse-fur and wrinkled skin phenotype. Of the 38 progeny produced in this control cross B, 14 were male, and of these, 8 were phenotypically normal and 6 were mutant, as determined by the 20 presence of wrinkled skin and abnormal fur. In the test cross with F1 mouse #213, of the 35 progeny produced in this cross, all eleven of the male progeny were phenotypically normal, clearly showing the genotyping of F1 mouse #213 as a germline transmitted gene corrected homozygous normal female. As another independent test to determine if the normal gene corrected allele in mouse #16 could be transmitted through the germline, mouse #16 was mated with homozygous (spf-ash/spf-ash) mutant 25 females. In this cross if mouse #16 does not transmit a normal allele, the resultant progeny will either be hemizygous (spf-ash/Y) mutant males or homozygous (spf-ash/spf-ash) mutant females, both of which are phenotypically mutant. However if the mouse allele is transmitted through the germline, heterozygous (spf-ash/+) females that are phenotypically normal will be produced. When mouse #16 was bred with homozygous (spf-ash/spf-ash) mutant females, two litters were produced that consisted 30 of a total of 5 hemizygous (spf-ash/Y) mutant males, 7 homozygous (spf-ash/spf-ash) mutant females, and 1 phenotypically normal female (mouse #1014). Pictures of representative mice from these crosses are shown in Figure 23. The production of the phenotypically normal female mouse provides direct genetic evidence that mouse #16 contains a normal gene corrected OTC allele that is germline transmissable. 76 WO 99/60108 PCTIUS99/10731 Although the present invention has been described in some detail by way of illustration for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the claims. 77

Claims (31)

1. A non-human mammal comprising a modified endogenous gene, wherein said endogenous gene is selected from the group consisting of a gene or sequence encoding an ion-channel, a G 5 protein coupled receptor (GPCR), an immunoglobulin, a growth factor, an enzyme, or a milk protein.

2. A mammal according to claim 1 wherein said mammal is a farm animal.

3. A mammal according to claim 2 wherein said farm animal is selected from the group consisting of cattle, sheep, pigs, horses and goats. 10

4. A mammal according to claim 1 wherein said mammal is selected from the group consisting of mice, rats, rabbits , guinea pigs, hamsters and gerbils.

5. A mammal according to claim 1 wherein said milk protein gene is a lactoglobulin gene.

6. A mammal according to claim 5 wherein said lactoglobulin gene is the "-lactoglobulin gene or the $-lactoglobulin gene. 15

7. A mammal according to claim 6 wherein said modified "-lactoglobulin gene or $-lactoglobulin gene does not encode any phenylalanine residues.

8. A mammal according to claim 1 wherein said endogenous gene is disrupted by deletion of at least one nucleotide.

9. A mammal according to claim 1 wherein said endogenous gene is disrupted by an insertion 20 sequence.

10. A mammal according to claim 9 wherein said insertion sequence is a polylinker sequence.

11. A mammal according to claim 9 wherein said insertion sequence is a reporter gene.

12. A mammal according to claim 11 wherein said reporter gene is selected from the group consisting of a luciferase gene, a P-galactosidase gene and green fluorescent protein (GFP), 78 WO 99/60108 PCT/US99/10731 blue fluorescent protein (BFP), red fluorescent protein (RFP) and yellow fluorescent protein (YFP).

13. A mammal according to claim 9 wherein said insertion sequence is selected from the group consisting of a gene encoding human lysozyme, human growth hormone, human serum 5 albumin, human globin, a human immunoglobulin, and a human enzyme.

14. A mammal according to claim 12 wherein said human enzyme is a-1 antitrypsin.

15. A mammal according to claim 12 wherein said human enzyme is anti-thrombin Ill.

16. A mammal according to claim 12 wherein said human enzyme gene does not encode any phenylalanine residues. 10

17. A mammal according to claim 9 wherein said insertion sequence is selected from the group consisting of a human gene under control of its endogenous promoter, a modified endogenous regulatory element for an endogenous gene, a transcriptional regulation cassette and a dimerizing sequence.

18. A mammal according to claim 17 wherein said endogenous regulatory element is disrupted 15 by deletion of at least one nucleotide.

19. A mammal according to claim 17 wherein said regulatory element is disrupted by an insertion sequence.

20. A mammal according to claim 1 wherein said enzyme is a sugar transferase enzyme.

21. A mammal according to claim 20 wherein said sugar transferase enzyme is "-galactosyl 20 transferase.

22. A mammal according to claim 21 wherein said "-galactosyl transferase gene is disrupted by deletion of at least one nucleotide.

23. A mammal according to claim 21 wherein said "-galactosyl transferase gene is disrupted by an insertion sequence. 79 WO 99/60108 PCT/US99/10731

24. A mammal according to claim 23 wherein said insertion sequence is a hormone receptor gene.

25. A mammal according to claim 23 wherein said insertion sequence is a viral receptor gene.

26. A mammal according to claim 23 wherein said insertion sequence is a G-protein coupled 5 receptor gene.

27. A primate comprising a modified endogenous gene.

28. A primate according to claim 27 wherein said endogenous gene is disrupted by deletion of at least one nucleotide.

29. A primate according to claim 27 wherein said endogenous gene is disrupted by an insertion 10 sequence.

30. A primate according to claim 29 wherein said insertion sequence is a human therapeutic gene.

31. A primate according to claim 29 wherein said insertion sequence is a human antibody gene. 80