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WO1993023553A1 - Procede de production d'organismes transgeniques par liaison in vivo de regions codantes et de regulation - Google Patents

Procede de production d'organismes transgeniques par liaison in vivo de regions codantes et de regulation Download PDF

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Publication number
WO1993023553A1
WO1993023553A1 PCT/US1993/004773 US9304773W WO9323553A1 WO 1993023553 A1 WO1993023553 A1 WO 1993023553A1 US 9304773 W US9304773 W US 9304773W WO 9323553 A1 WO9323553 A1 WO 9323553A1
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Prior art keywords
cell
operably linked
region
coding region
regulatory region
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Inventor
Samuel Wadsworth
Paul J. Leibowitz
Benjamin Synder
Patrice Milos
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Exemplar Corp
rEVO Biologics Inc
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Exemplar Corp
TSI Corp
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Publication of WO1993023553A1 publication Critical patent/WO1993023553A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • a method of treating cells to add a desired characteristic which may be predetermined is provided.
  • a regulatory region and a coding region that are free of operable linkage are introduced into a cell. These regions are constructed and arranged such that they can join in vivo to form an operably linked unit.
  • the regulatory region and coding region are permitted to join in vivo to form such an operably linked unit, which aids in obtaining said desired characteristic.
  • transgenic non-human organisms which have added a desired characteristic by the method of this invention, are provided.
  • Figure la depicts the ligation of blunt ends on the downstream end of a CMV promoter region and the upstream end of a ⁇ -galactosidase coding region where the promoter region and the ⁇ -galactosidase coding region are on different DNA segments.
  • Figure lb depicts the ligation of blunt ends on the downstream end of a CMV promoter region and the upstream end of a ⁇ -galactosidase coding region where the promoter region and the ⁇ -galactosidase coding region are on the same DNA segment-
  • Figure 8 depicts the coding regions for the myc and ras oneogenes.
  • join means any process whereby the regulatory region and the coding region are physically connected so as to form an operably linked unit. Such a process includes joining of double stranded blunt DNA ends, joining of single stranded cohesive DNA ends, recombination between homologous regions of DNA, and non-homologous recombination. In vivo joining is able to occur under ordinary conditions of cell growth. Any temperature that is not destructive to the cell can be used. The ⁇ -n vivo joining is believed to occur substantially instantaneously.
  • the regulatory region and coding region are constructed .in vitro such that the downstream end of the regulatory region and the upstream end of the coding region have blunt DNA ends.
  • the joining of the two DNA regions can be accomplished by generating complementary sequences on the ends of each of the regions, so that they can recombine into an operably linked unit when introduced together in vivo.
  • the complementary sequences are of sufficient length and sequence content to promote association of the regulatory region with the coding region in vivo.
  • the generation of complementary sequences can be accomplished in many ways. For example, a restriction enzyme may be used that makes staggered cuts to generate short, complementary single-stranded cohesive ends. This complementarity allows the cohesive ends to anneal by base pairing.
  • the single stranded DNA ends have a high G+C content because such ends favor a more stable association.
  • a similar number of nucleotides can be removed from the regulatory region and coding region molecules, thereby revealing single stranded cohesive ends that associate with each other in an intracellular environment.
  • Single stranded sticky ends are generated in a homologous sequence region, depicted by the stippled box between the Xhol and NotI restriction endonuclease sites on the downstream end of a CMV promoter region, and in a homologous sequence region between the Xhol and NotI sites on the upstream end of a ⁇ -galactosidase coding region, in the presence of exonuclease III.
  • These sticky ends anneal to form an operably linked unit.
  • the joining of the regulatory region and the coding region is accomplished by constructing in vitro a regulatory region with a sequence at its downstream end that is homologous to a sequence at the upstream end of the coding region, such that the homologous regions are able to undergo homologous recombination with each other in vivo.
  • the sequence region should be of a length that is sufficient to permit i-n vivo homologous recombination to occur. See Figure 4 in which a homologous sequence region, depicted by the stippled box between the Xhol and NotI restriction endonuclease sites on -li ⁇
  • the DNA molecules can be introduced into the cell by any process which results in DNA uptake including microinjection, electroporation, or retroviral infection.
  • Introduction of the DNA into a cell includes introduction into cultured cells, tissues or the whole or part of a living organism.
  • the two molecules can be introduced at the same time or separately. If they are introduced separately, they must be introduced within a close enough time span such that in vivo joining of the two molecules is able to occur. Preferably, the two molecules are introduced into the cell at the same time.
  • Incorporation may result in integration of a single operably linked unit or in the formation of conca emer ⁇ .
  • concatemer it is meant a sequence of DNA consisting of a series of unit lengths repeated in tandem. Concatemers may be very long in that the unit length sequence can be repeated over and over. Concatemers may be formed in head to tail, or tail to tail, or head to head orientation. The formation of concatenates in the genome can be monitored by cleavage with a panel of restriction endonucleases. If an enzyme is used which has a single site within the linear molecule, head to tail concatenates are seen as unit length DNA fragments.
  • the extent of concatenation is estimated from the ratio of hybridization intensity of the unit length fragment to that of terminal DNA fragments resulting from cutting at genomic sites that are flanking the insertion site.
  • Tail to tail and head to head concatenates and single unit insertions can be distinguished by their restriction patterns.
  • integration may occur at only one chromosomal site or at multiple sites.
  • the .in vivo formed operably linked unit can be analyzed to confirm its structure. Restriction endonuclease analysis is used to obtain information regarding its gross structure. Enzymes are used that do not cleave within the introduced DNA molecules but do cleave in the flanking genomic DNA. The number of such DNA fragments that hybridize with a probe specific for the introduced DNA indicates the number of integration sites. Restriction endonuclease analysis is also used to obtain a preliminary analysis of the molecular structure of the integrated DNA.
  • Assays include histochemical assays, immunohistochemical assays, enzymatic assays, protein purification, in situ hybridization methods in whole organisms, tissue sections, cell homogenates or single cells, RNA hybridization and RNAse protection assays. If the gene product of one of the coding regions is closely related to an endogenous gene product such that antibody cross-reactivity and in situ hybridization probe cross-reactivity are high, a determination of expression of such a coding sequence preferably is obtained by RNAse protection, specific antibody reaction, or PCR amplification. Where multiple regulatory regions are introduced e.g. , where the different regulatory regions direct expression in different tissues, expression of the coding region (or coding regions) is assayed in the expected range of tissues. Participation of the different regulatory regions in expression is assayed by PCR amplification using primers specific to each promoter in combination with primers specific to each coding sequence.
  • This invention also includes a method for changing the genotype of a non-human organism to produce an abnormal condition in the organism which simulates a condition sometimes occurring in nature.
  • the term simulates a condition sometimes occurring in nature means that a condition is created in the organism which is similar to a condition that might occur in nature as a result of environmental events and/or other stimuli acting on the organism.
  • abnormal condition it is meant a condition which differs from the normal healthy condition of an organism.
  • Such abnormal conditions include the acquisition of a characteristic which results in a diseased condition in the organism. Examples of a diseased condition include cancer, immune system deficiency, inflammatory disease, neurodegenerative disease, diabetes, thrombus formation and inherited metabolic disorders.
  • An abnormal condition also includes a condition which increases the ability of an organism to withstand organ transplantation.
  • Models can be produced for: cancer through the expression of viral proteins, activated oncogenes, and modulation of the expression of anti-oncogenes (Hanahan (1989) Science 246: 1265-1274; Bailleul et al. (1990) Cell: 62: 697-708; Haupt et al. (1991), Cell 65: 753-763; Lavrequisite et al. (1989) Mol ⁇ Cell. Biol. 9: 3982-3991; Pattengale et al. (1989) Am. J. Path. 135: 39-61; Donehower et al.
  • This invention further includes a method for changing the genotype of an organism derived at least partially from a treated cell so as to obtain a desired characteristic, which may be predetermined, in the organism.
  • a desired characteristic in an animal include enhanced milk production, disease resistance, growth enhancement, enhanced nutritional value and production of a desired protein.
  • a desired characteristic in a plant include disease resistance, altered ripening characteristics, growth enhancement, enhanced nutritional value and production of a desired protein.
  • Genes involved in disease resistance include gene products that counteract viral infections. (Muller and Brem (1991) Experienta 47: 923-934).
  • Enhanced milk production is meant to include increased levels of milk, enhanced and/or novel nutritional value of milk, and milk containing various pharmaceutical products.
  • compositions that can be produced in milk include tissue plasminogen activator and ⁇ -1-antitrypsin (Wright et al. (1991) Bio/technology 9_: 830-834; Ebert et al. (1991) Bio/technology 9: 835-843).
  • Growth enhancement is meant to include faster growth, increased body size and increased litter size.
  • Genes involved in growth enhancement include the gene for growth hormone. (Palmiter et al. (1982) Nature 300: 611-615); Palmiter et al. (1983) Science 222: 809-814).
  • Enhanced nutritional value is meant to include greater amounts of a nutrient, novel nutrients, the absence of certain nutrients and leaner meat. Genes involved in enhanced nutritional value include the ski gene which results in leaner meat.
  • a desired protein means a protein that bestows a desired trait on the organism in which it is produced or a protein which when isolated from the organism is desirable for uses outside of the organism.
  • the desired protein may be produced in a specific tissue, a subset of tissues or in a wide range of tissues, depending upon the use for which production of the protein is desired. Production of the protein also can be designed so that it is made at a desired time or times during the life of the organism.
  • desired proteins include therapeutic proteins and proteins which correct an abnormal condition in the organism. Correction of an abnormal condition includes gene therapy.
  • Gene therapy which has a wide variety of applications for mammals and in particular for humans, can be mediated by the introduction of the DNA encoding the therapeutic product by at least three different routes.
  • Cells that can potentially be used are keratinocytes (Green, 1991, Sci. Am. 265, 96-102), lymphocytes Culver et al., 1991, PNAS USA 88, 3155-3159), bone marrow cells (Williams, 1990, Hum. Gene Ther .
  • Viruses that are current candidates for incorporation and introduction of therapeutic DNA molecules include retroviruses, and adenoviruses that can infect human cells (Culver et al., 1991, PNAS USA 88, 3155-3159; Wang et al, 1991, Adv. Exp. Med. Biol. 309, 61-66; Wilson et al. 1992, Science 244, 1344-1346; Rosenfeld et al .
  • the abnormal condition or obtaining the desired characteristic can be facilitated by, or even dependent upon, selection of a particular regulatory region.
  • the regulatory region may be preselected for its expected activity in a particular tissue or for its ability to be regulated by an event such as neoplastic transformation. Preselection will depend upon the characteristic or abnormal condition that is desired.
  • this invention also provides for cultures of cell lines that can be the source for desired products as a result of containing the operably linked unit stably incorporated into the genome of the cells in the cell line.
  • This invention also includes breeding an organism, or an offspring of an organism, which was produced by the methods of this invention. Breeding is performed by normal and customary techniques.
  • This invention also includes a transgenic non-human organism having added a desired characteristic from having incorporated into the genome of at least some ceils an operably linked unit resulting from the method described above, a cell derived from a somatic cell from such a transgenic organism, and a cell line derived from such a somatic ceil.
  • This invention further includes a cell or a cell line having added a desired characteristic from having incorporated into its genome an operably linked regulatory region and coding region by any of the methods described above.
  • Restriction endonucleases are obtained from conventional commercial sources such as New England Biolabs (Beverly, MA) , Promega Biological Research Products (Madison, WI) and Stratagene (LaJolla, CA) .
  • Radioactive materials are obtained from conventional commercial sources such as Dupont/NEN or Amersham.
  • Custom-designed oligonucleotides for PCR are obtained from any of several commercial providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Animals suitable for transgenic experiments are obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY) and Harlan Sprague Dawley (Indianapolis, IN). Swiss Webster female mice are used for embryo retrieval and transfer. B6D2F., males are used for mating and vasectomized Swiss Webster studs are used to stimulate pseudopregnancy. Vasectomized mice and rats are obtained from the supplier.
  • DNA clones are cleaved with appropriate enzymes and the DNA fragments are electrophoresed on 1% agarose gels in TBE buffer.
  • the DNA bands are visualized by staining with ethidium bromide, excised, and placed in dialysis bags containing 0.3M sodium acetate, pH 7.0.
  • DNA is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of ethanol .
  • the DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D column.
  • the column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer.
  • the DNA solutions are cleaved with appropriate enzymes and the DNA fragments are electrophor
  • DNA concentrations are measured by absorption at 260 nm in a UV spectrophotomer. For microinjection, the DNA concentration is.adjusted to 3 ⁇ g/ml in 5 mM tris, pH 7.4 and 0.1 mM EDTA.
  • mice six weeks of age, are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma), followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma) .
  • Females are placed with males immediately after hCG injection. Twenty-one hours after hCG, the mated females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma) .
  • BSA bovine serum albumin
  • hyaluronidase (1 mg/ml).
  • Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5°C incubator with a humidified atmosphere at 5% C0 2 , 95% air until the time of injection.
  • EBSS Earle's balanced salt solution containing 0.5% BSA
  • transgenic rats The procedures for generating transgenic rats are similar to those for mice. Sprague Dawley rats are used for all procedures. Thirty day-old female rats receive a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female is placed with proven male. Also at this time, 40-80 day old females are placed in cages with vasectomized males. These will provide the recipient females for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer.
  • Donor females that have mated are sacrificed (C0 2 asphyxiation) and their oviducts removed, placed in DPBS (Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSS (Earle' ⁇ balanced salt solution) containing 0.5% BSA in a 37.5°C incubator until the time of microinjection.
  • DPBS Dynabecco's phosphate buffered saline
  • hyaluronidase (1 mg/ml).
  • the embryos are then washed and placed in EBSS (Earle' ⁇ balanced salt solution) containing 0.5% BSA in a 37.5°C incubator until the time of microinjection.
  • the live embryos are moved to DPBS for transfer into recipient females.
  • the recipient females are anesthetized with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip) .
  • a dorsal midline incision is made through the skin and the ovary and the oviduct is exposed by an incision through the muscle layer directly over the ovary.
  • the ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum. Approximately 10-12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the recipient females are housed singly.
  • Tail samples (1-2 cm) are removed from three week old animals. DNA is prepared and analyzed by both Southern blot and PCR to detect transgenic founder (F Q ) animals and the progeny (F, and F 2 ) . -23-
  • An organism is identified as a potential transgenic by taking a sample of the organism for DNA extraction and hybridization analysis with a probe complementary to the transgene of interest.
  • DNA extracted from the organism can be subjected to PCR analysis using PCR primers complementary to the transgene of interest.
  • Evaluation of the gross structure of the operably linked unit in transgenic non-human organisms is carried out by conventional restriction endonuclease analysis as discussed above in order to reveal the number of integration sites.
  • a preliminary evaluation of the molecular structure of the junction of monomers of injected DNA molecules within concatenates is carried out by restriction endonuclease digestion.
  • restriction endonuclease digestion For example, in the case of concatenates formed after Smal cleavage of plasmid pCMV ⁇ , cleavage with Xhol and Pvul in a head to tail concatenate yields a 500 base pair fragment. See Figure 5 which depicts plasmid pCMV ⁇ with the relevant restriction endonuclease sites noted. Removal of as few as about 50 nucleotides from the injected DNA molecule by a non-specific nuclease during concatenation is readily detected by gel electrophoresis.
  • variation in the length of the junction fragment indicates the incorporation of more than one processed monomer into the concatenate.
  • Uniform junction fragment length of the expected size indicates that only non-processed monomers are present in the concatenate.
  • Uniform junction fragment length of less than the expected size indicates that only one class of processed monomers is present in the concatenate.
  • a detailed evaluation of the molecular structure of the junction fragment is carried out through PCR amplification and sequencing.
  • Figure 6 in which an upstream and a downstream PCR primer are used to amplify a junction fragment from the genome that is generated as a result of joining a CMV promoter region with a ⁇ -galactosidase coding region.
  • the PCR product is then cleaved, for example, with Xhol and Pvul (see Figure 6), and is subcloned, for example, into a pBC plasmid vector (Stratagene, LaJolla, CA) for sequencing.
  • a pBC plasmid vector Stratagene, LaJolla, CA
  • the plasmid, pCMV ⁇ (Clontech, Palo Alto, CA) , contains the promoter region from the dominant immediate early gene from human cytomegaloviru ⁇ (1.9kb mRNA; Boshart et al. (1985), Cell 4.:521-530) functionally linked to the ⁇ -galactosidase gene from the E. coli lactose operon.
  • the CMV promoter is expressed in a wide variety of tissues in transgenic animals (Schmidt et al . (1990), Mol. Cell. Biol. ] ):4406-4411; Furth et al . (1991), Nuc. Acids Res.
  • cells in a transgenic animal that express this construct are identified by assaying for ⁇ -galactosidase enzyme activity.
  • a common and simple ⁇ -galactosidase assay employs the chromogenic substrate, X-gal, producing an intense blue color. Because of the ease of this assay, this DNA molecule is used to exemplify the degree of success of the process.
  • a site for the blunt end cutting restriction enzyme, Smal is positioned such that it lies between the promoter and the initiator methionine codon of the ⁇ -galactosidase coding sequence. Cleavage at this Smal site inactivates the ⁇ -galactosidase gene by separating the promoter from the ⁇ -galactosidase coding sequence. Thus, transcription from the CMV promoter does not proceed through the ⁇ -galactosida ⁇ e coding sequence.
  • Smal-cleaved pCMV ⁇ DNA prepared using the methods described in this application, is injected into pronuclei of fertilized mouse eggs and further used to generate transgenic animals following the procedures described in this application.
  • a Sail site is centrally located within the injected linear molecule (see Figure 5), and head to tail concatenates are therefore seen as unit length DNA fragments.
  • the extent of concatenation is estimated from the ratio of hybridization intensity of the unit length fragment to that of terminal DNA fragments resulting from cutting at the genomic Sail sites flanking the insertion site.
  • Formation of head to head or tail to tail concatenates is monitored by cleavage with EcoRI which cuts asymmetrically within the linear molecule that is originally injected. Single site insertions are revealed by the presence of only two EcoRI DNA fragments, neither of which is unit length.
  • Head to tail concatenate ⁇ regenerate the functional expression cassette while other concatenates and single site insertions of either promoter or coding sequence fragments do not.
  • tissues from transgenic animals are assayed for ⁇ -galactosidase activity as described in Tan (1991).
  • a blunt-cutting restriction enzyme is used to sever the promoter sequence from the coding sequence.
  • a restriction endonuclease that leaves sticky ends are similarly used. The choice of enzyme is dictated by the exact structure of the DNA molecules used in the production of transgenic animals.
  • the promoter region of the rat ⁇ -tropomyosin gene is included in a 4.2 kb BamHI to Apal DNA segment (Ruiz-Opazo et al. J. Biol. Chem. (1990) 265:9555-9562). See Figure 7 in which this 4.2 kb fragment with some of it ⁇ restriction endonuclease site ⁇ i ⁇ depicted. The arrow in the figure -27-
  • the Apal site indicated in the figure separates the promoter region from the protein coding region.
  • the Apal site was altered through conventional molecular genetic techniques and synthetic oligonucleotide linkers to generate a Sail site.
  • the ⁇ -galactosidase coding region from the pCMV ⁇ clone shown in Figure 5 is removed as an Xhol to Sail fragment.
  • the four nucleotides of the 5' sticky end of the Sail site, 5'-TCGA-3', are complementary to the 5' sticky ends of the Xhol site, 3'-AGCT-5'.
  • the ⁇ -tropomyosin promoter is expre ⁇ ed in a variety of tissues including striated muscle, smooth muscle, fibroblasts, and brain (Goodwin et al. (1991) J. Biol. Chem. 266: 8408-8415).
  • the ⁇ -galactosidase protein i ⁇ readily assayed in cells and tissues by providing the recombinant cell or tissue with any of a variety of sub ⁇ trates for the enzyme.
  • the preferable sub ⁇ trate is the compound X-gal, which will produce an intense blue reaction product in the presence of the enzyme.
  • the ⁇ -tropomyosin promoter DNA fragment described above is used as the ⁇ eparate promoter DNA fragment.
  • the oncogene ⁇ used are ras and myc (Pattengale et al. (1989) Am. J. Path. 135: 39-61).
  • the coding regions of these oncogene ⁇ are contained on the DNA fragment ⁇ ⁇ hown in Figure 8.
  • the ras oncogene is excised as a Smal fragment and the myc oncogene is excised as an Xbal to EcoRI fragment.
  • the blunt ends of the Smal cut DNA fragment ⁇ are left untreated while the sticky ends of restriction enzyme cleaved DNA fragments are filled in with DNA polymerase (Sambrook et al.
  • This invention is employed to provide an even simpler a ⁇ ay for activity of the ⁇ -tropomyo ⁇ in promoter in ti ⁇ ue ⁇ of the animal line in which tumor formation i ⁇ being monitored. This is accomplished by causing ⁇ -galactosida ⁇ e expre ⁇ ion to be brought under control of the ⁇ -tropomyosin promoter in the same tissue ⁇ in which the oncogene ⁇ equence ⁇ are controlled by the ⁇ -tropomyo ⁇ in promoter. Thi ⁇ is accomplished by including the above noted DNA fragment encoding the ⁇ -galactosida ⁇ e coding ⁇ equence in the mixture of DNA molecules injected into the fertilized egg.
  • the ⁇ -tropomyosin promoter thus is controlling expression of two oncogenes as well as the ea ⁇ ily assayable ⁇ -galactosidase gene in a given set of tissues in a given line of transgenic animal ⁇ .
  • the simple ⁇ -galactosida ⁇ e assay takes the place of the more laborious and expensive in situ hybridization.
  • a further extension of this application of the invention involves the use of one or more promoters in addition to the ⁇ -tropomyosin promoter in the mixture of DNA molecules injected into fertilized eggs.
  • the spectrum of tissues in which the additional promoters are active similarly increases the spectrum of tissues in which tumors are produced, thereby making the transgenic animals more useful.

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Abstract

L'invention concerne un procédé qui consiste à introduire dans une cellule une région de régulation non liée fonctionnellement et une région codante, ces dernières étant construites de manière à pouvoir se lier in vivo afin de former une unité liée fonctionnellement. Avant l'introduction dans la cellule, les extrémités de la région de régulation et de la région codante sont construites de manière à contenir, par exemple, des extrémités franches de façon à permettre la ligature des extrémités franches, des bouts collants simple brin complémentaires de manière à permettre l'annelage entre les brins, ou des régions à séquence homologue de façon que la recombinaison homologue soit possible. Des organismes, des cellules et des lignées cellulaires transgéniques sont également décrits.
PCT/US1993/004773 1992-05-19 1993-05-19 Procede de production d'organismes transgeniques par liaison in vivo de regions codantes et de regulation Ceased WO1993023553A1 (fr)

Applications Claiming Priority (2)

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US88552092A 1992-05-19 1992-05-19
US07/885,520 1992-05-19

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WO1993023553A1 true WO1993023553A1 (fr) 1993-11-25

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AU (1) AU4383293A (fr)
WO (1) WO1993023553A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357127A1 (fr) * 1988-08-16 1990-03-07 Gist-Brocades N.V. Remplacement de gènes comme règle de construction de souches d'aspergillus
WO1991009955A1 (fr) * 1989-12-22 1991-07-11 Applied Research Systems Ars Holding N.V. Modification de l'expression de genes endogenes a l'aide d'un element regulateur
WO1992003917A1 (fr) * 1990-08-29 1992-03-19 Genpharm International Recombinaison homologue dans des cellules de mammiferes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0357127A1 (fr) * 1988-08-16 1990-03-07 Gist-Brocades N.V. Remplacement de gènes comme règle de construction de souches d'aspergillus
WO1991009955A1 (fr) * 1989-12-22 1991-07-11 Applied Research Systems Ars Holding N.V. Modification de l'expression de genes endogenes a l'aide d'un element regulateur
WO1992003917A1 (fr) * 1990-08-29 1992-03-19 Genpharm International Recombinaison homologue dans des cellules de mammiferes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANNUAL REVIEW OF GENETICS vol. 23, 1989, PALO ALTO, USA pages 199 - 225 BOLLAG, R. J. ET AL. 'Homologous recombination in mammalian cells' *
GENETICS vol. 111, October 1985, pages 375 - 388 AYARES, D. ET AL. 'Homologous recombination between autonomously replicating plasmids in mammalian cells' *
JOURNAL OF VIROLOGY vol. 62, no. 6, June 1988, pages 2191 - 2195 VAN ZIJL, M. ET AL. 'Regeneration of Herpesviruses from molecularly cloned subgenomic fragments' *
MOLECULAR AND CELLULAR BIOLOGY vol. 5, no. 1, January 1985, pages 59 - 69 FOLGER, K. R. ET AL. 'Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei' *

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