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WO2001018225A1 - Constructions de ciblage et animaux transgeniques produits avec ces constructions - Google Patents

Constructions de ciblage et animaux transgeniques produits avec ces constructions Download PDF

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Publication number
WO2001018225A1
WO2001018225A1 PCT/US1999/030078 US9930078W WO0118225A1 WO 2001018225 A1 WO2001018225 A1 WO 2001018225A1 US 9930078 W US9930078 W US 9930078W WO 0118225 A1 WO0118225 A1 WO 0118225A1
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vector
gene
sequences
mammal
transgenic
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Ning Zhang
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Xenogen Corp
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Xenogen Corp
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • 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
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests

Definitions

  • This invention is in the field of molecular biology and medicine. More specifically, it relates to novel vector constructs and methods of use thereof for introducing heterologous polynucleotides into a host cell. Further, the invention relates to vector constructs and methods of use thereof to generate transgenic organisms, particularly transgenic mice.
  • mice geneticists have succeeded in creating transgenic animals by manipulating the genes of developing embryos and introducing foreign genes into these embryos. Once these genes have integrated into the genome of the recipient embryo, the resulting embryos or adult animals can be analyzed to determine the function of the gene.
  • transgenic mice have been generated through DNA microinjection approach. Such an approach leads to the creation of "founder" mice having at least one copy of the transgene randomly integrated into the genome. Because neither the copy number nor the integration sites can be controled, transgenic mice generated by this method are genetically different from each other. The expression of transgenes in such transgenic mice are not uniform because of, for example, the difference in copy numbers of the transgene. Furthermore, chromosomal location of the transgene often affects the expression level of the transgene. For most of in vivo studies, particularly ones where it is desirable to compare levels of gene expression across different animals, it is important to have the mice with little or no genetic variation (i.e., isogenic mice) in order to reduce the systematic error.
  • the integration disrupts the function of the target gene allows for examination of the phenotype resulting from the disruption of the gene.
  • the present invention solves this and other problems by providing transgenic animals in which at least one single-copy, non-essential gene is replaced with a reporter expression cassette, for example, a luciferase gene operably linked to a heterologous promoter.
  • the present invention provides novel methods and vector constructs useful for the generation of transgenic animals.
  • the invention further includes methods of using these animals.
  • transgenic animals described herein are useful, for example, when studying in vivo regulation of selected genes. Also described herein are methods of generating populations of substantially isogenic transgenic animals, as well as, vectors useful in these methods.
  • the subject invention is directed to a transgenic, non-human mammal, for example, a rodent such as a mouse.
  • the mammal comprises at least one single-copy, non-essential gene in its genome, wherein (i) at least a portion of at least one single-copy, non-essential gene is replaced by polynucleotide sequences heterologous to the gene, and (ii) the polynucleotide sequences comprise a first expression cassette which has been introduced into the mammal or an ancestor of the mammal, at an embryonic stage.
  • the first expression cassette typically comprises a first selectable marker, a first transcriptional promoter element heterologous to the gene, and light-generating protein coding sequences.
  • the light-generating protein coding sequences are operably linked to the promoter element.
  • the single-copy, non-essential gene may be selected, for example, from the group consisting of vitronectin,/o55, and galactin 3 and the first selectable marker may be selected from the group consisting of neomycin phosphotransferase II, xanthine- guanine phosphonbosyltransferase, hygromycin-B-phosphotransferase, chloramphenicol acetyltransferase, and adeninephosphoribosyl transferase.
  • the first transcriptional promoter element is an inducible promoter, a repressible promoter, or a constitutive promoter, and may be selected from the group consisting of VEGF, VEGFR, and TIE2.
  • the transgenic, non-human mammal described above comprises a second single-copy, non-essential gene in its genome, wherein (i) at least a portion of the second single-copy, non-essential gene is replaced by polynucleotide sequences heterologous to the second gene, and (ii) the polynucleotide sequences comprise a second expression cassette which has been introduced into the mammal or an ancestor of the mammal, at an embryonic stage.
  • the second expression cassette typcially comprises a second selectable marker, a second transcriptional promoter element heterologous to the second gene, and light generating protein coding sequences.
  • the light generating protein coding sequences are operably linked to the promoter element.
  • the first and second transcriptional promoter elements and selectable markers may be the same or different and the light generating protein in the first expression cassette can produce a different color of light relative to the light generating protein in the second expression cassette.
  • the invention is directed to a method of producing a transgenic, non-human mammal, such as a mouse.
  • the mammal has at least one single- copy, non-essential gene in its genome.
  • the method comprises transfecting an embryonic stem cell of the mammal with a linear vector comprising
  • a first selectable marker and a reporter expression cassette comprising a transcriptional promoter element operably linked to a light generating protein coding sequence
  • the offspring is capable of germline transmission of the reporter expression cassette and the method may further comprise breeding the offspring with a mammal which is substantially isogenic with the embryonic stem cells, such that the breeding yields transgenic FI offspring carrying the reporter cassette.
  • the method comprises breeding the first FI offspring carrying the reporter cassette with a second FI offspring carrying the reporter cassette, wherein the breeding yields transgenic F2 offspring carrying the reporter cassette.
  • the embryonic stems cells may be derived from a mouse having a dark coat color, the mammal substantially isogenic with the embryonic stem cells may have a light coat color, and/or the F2 offspring carrying the reporter cassette may have a light coat color.
  • the embryonic stems cells are derived from a C57BL 6 mouse having a dark coat color, and the mammal substantially isogenic with the embryonic stem cells is a C57BL/6-Tyr C2j/+ mouse having a light coat color.
  • the subject invention is directed to a vector for use in generating a transgenic non-human mammal, for example, a rodent such as a mouse.
  • the mammal has at least one single-copy, non-essential gene in its genome.
  • the vector comprises (a) a first selectable marker and a reporter expression cassette, the reporter expression cassette comprising a transcriptional promoter element operably linked to a light generating protein coding sequence, and (b) targeting polynucleotide sequences homologous to a single-copy, non- essential gene in the mammal's genome, the targeting polynucleotide sequences flanking (a), wherein (i) the length of the targeting polynucleotide sequences are sufficient to facilitate homologous recombination between the vector and the single-copy, non- essential gene, and (ii) the transcriptional promoter element is heterologous to the single- copy, non-essential gene.
  • the first selectable marker provides a positive selection and may be selected from the group consisting of neomycin phosphotransferase II, xanthine-guanine phosphoribosyltransferase, hygromycin-B-phosphotransferase, chloramphenicol acetyltransferase, and adeninephosphoribosyl transferase.
  • the transcriptional promoter element may be an inducible promoter, a repressible promoter, or a constitutive promoter, and may be selected from the group consisting of VEGF, VEGFR, and TIE2.
  • the vector further comprises a second selectable marker and at least one target polynucleotide sequence is located between the second selectable marker and the first selectable marker.
  • the second selectable marker may provide a negative selection and may be selected from the group consisting of adenosine deaminase, thymidine kinase, and dihydrofolate reductase.
  • the vectors described above may be circular and may contain at least one restriction site whose cleavage results in a linear vector having the following arrangement of elements: target polynucleotide sequence - (a) - targeting polynucleotide sequences or target polynucleotide sequence - (a) - targeting polynucleotide sequences - (second selectable marker).
  • the coding sequences of the reporter expression cassette present in the vector may comprise codons that are optimal for expression in a host system into which the expression cassette is to be introduced. Additionally, the targeting polynucleotide sequences from single-copy, non-essential genes may be selected from the group consisting of vitronectin,/o5_ , and galactin 3.
  • the light-generating protein in the mammals and methods described above may be derived from either procaryotic or eucaryotic sources and, in particularly preferred embodiments, the light generating protein is a luciferase.
  • Figure 1 is a schematic depicting construction of the pTK53 vector.
  • Polynucleotides encoding PGK-P, Neo and TK and 5' and 3' linkers are introduced into a pKS backbone to produce the vector designated pTK53.
  • Figure 2 is schematic depicting construction of the pTK-LucR and pTK-LucYG vectors.
  • pTK-LucR a polynucleotide encoding LucR is introduced into pTK53.
  • the pTK-LucR construct contains the PGK-P gene, a neomycin (Neo r ) gene, a thymidine kinase (TK) gene and sequence encoding red luciferase (Luc-R).
  • TK-LucYG a polynucleotide encoding LucYG is introduced into pTK53.
  • the pTK- LucYG construct contains the PGK-P gene, a neomycin (Neo r ) gene, a thymidine kinase (TK) gene and a sequence encoding yellow-green luciferase (Luc-YG).
  • Figures 3 A is a schematic depicting the vector pTKLR-Vn. Sequences homologous to the vitronectin gene are inserted into pTK-LucR such that they flank the Neo r gene and the Luc-R coding sequence.
  • Figure 3B is a schematic depicting targeting of the linearized pTKLR-Vn vector to the vintronectin chromosomal locus. The VEGF promoter is cloned into the polylinkers between Neo and Luc-R. Upon homologous recombination, the Neo-VEGF-LucR transgene is inserted into the Vn gene.
  • (A) shows the targeting vector pTKLR-Vn
  • (B) shows the mouse vitronectin gene.
  • Neo - neomycin resistance encoding sequences In the figure, Neo - neomycin resistance encoding sequences; TK - thymidine kinase encoding sequences; LucR - red luciferase from pGL3Red (Dr. Christopher Contag, Stanford University, Stanford, CA). Regions bearing Vn gene translational start and stop codons are indicated with arrows. Poly(A) sequences are placed upstream of the polylinker to prevent or minimize read-through translation.
  • Figure 3C shows the nucleotide sequence of vitronectin (SEQ ID NO:38).
  • Figure 4A is a schematic depicting the vector pTKLG-Fos. Sequences homologous to the FosB gene are inserted into pTK-LucYG such that they flank the Neo r gene and the Luc-YG coding sequence.
  • Figure 4B shows the nucleotide sequence of FosB (SEQ ID NO:39).
  • Figure 5A is a schematic depicting targeting of the linearized pTKLG-Fos vector to the FosB chromosomal locus.
  • the VEGFR2 promoter is cloned into the polylinkers between Neo and Luc-YG.
  • the Neo-VEGFR2-LucYG transgene will be inserted into a sequence associated with production of FosB.
  • (A) shows the targeting vector
  • (B) shows the mouse target gene.
  • Neo - neomycin resistance encoding sequences TK - thymidine kinase encoding sequences
  • LucYG - yellow green luciferase from pGL3-control vector (Promega, Madison, WI).
  • Regions bearing FosB gene translational start and stop codons are indicated with arrows.
  • Poly(A) sequences are placed upstream of the polylinker to prevent or minimize read-through translation.
  • Figure 5B is a schematic depicting targeting of the linearized pTKLG-Fos vector to the FosB chromosomal locus.
  • the TIE2 promoter is cloned into the polylinkers between Neo and Luc-R. Upon homologous recombination, the Neo-Tie2-LucYG transgene is inserted into the FosB gene.
  • (A) shows the targeting vector
  • (B) shows the mouse target gene.
  • Neo - neomycin resistance encoding sequences TK - thymidine kinase encoding sequences
  • LucYG - yellow green luciferase from pGL3-control vector (Promega). Regions bearing FosB gene translational start and stop codons are indicated with arrows.
  • Poly(A) sequences are placed upstream of the polylinker to prevent or minimize read-through translation.
  • Figure 6 depicts PCR conditions for genomic screening for promoters useful in exemplary targeting constructs of the present invention.
  • Figure 7 depicts generation of targeted transgenic mice using the targeting vectors described herein.
  • Figure 8 depicts of schematic representation of Southern blot analysis of homologous DNA recombination between pTKLG-Fos targetting vector and the FosB gene.
  • Figure 9 depicts generation of targeted transgenic mice, using the targeting vectors described herein, and crosses using such transgenics as well as their offspring (FI, first generation; F2, second generation).
  • Figure 10 depicts crosses using transgenic mice of the present invention to generate dual luciferase transgenic mice. MODES FOR CARRYING OUTTHE INVENTION
  • nucleic acid molecule and “polynucleotide” are used interchangeably to and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides branched polynucleotides
  • plasmids vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • T thymine
  • the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • a “coding sequence” or a sequence which "encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • Other "control elements" may also be associated with a coding sequence.
  • a DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence.
  • "Encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide encoded by the sequence.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), translation enhancing sequences, and translation termination sequences.
  • Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
  • “Expression enhancing sequences” typically refer to control elements that improve transcription or translation of a polynucleotide relative to the expression level in the absence of such control elements (for example, promoters, promoter enhancers, enhancer elements, and translational enhancers (e.g., Shine and Delagarno sequences)).
  • “Purified polynucleotide” refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • a “heterologous sequence” as used herein is typically refers to either (i) a nucleic acid sequence that is not normally found in the cell or organism of interest, or (ii) a nucleic acid sequence introduced at a genomic site wherein the nucleic acid sequence does not normally occur in nature at that site.
  • a DNA sequence encoding a polypetide can be obtained from yeast and introduced into a bacterial cell. In this case the yeast DNA sequence is "heterologous" to the native DNA of the bacterial cell.
  • a promoter sequence from a Tie2 gene can be introduced into the genomic location of afosB gene. In this case the Tie2 promoter sequence is "heterologous" to the native fosB genomic sequence.
  • a "polypeptide” is used in it broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, for example ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is typically called a polypeptide or a protein.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter that is operably linked to a coding sequence e.g., a reporter expression cassette
  • the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Recombinant host cells “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting procaryotic microorganisms or eucaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
  • An "isolated polynucleotide” molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.
  • sequence identity also is known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more sequences can be compared by determining their "percent identity.”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences 5 divided by the length of the shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages 0 the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six).
  • the desired degrees of sequence identity are at least 80%, 85-90%, preferably 92%, more preferably 95%, and even more preferably 98% sequence identity to the reference sequence (i.e., the sequences of the present invention).
  • the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded- specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 85%-90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • Two nucleic acid fragments are considered to "selectively hybridize" as described herein.
  • the degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules.
  • a partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency.
  • the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a hybrid molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10- 14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe.
  • Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford; Washington, DC; IRL Press).
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions.
  • the selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
  • a “vector” is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • vector transfer vector means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • Nucleic acid expression vector or “expression cassette” refers to an assembly which is capable of directing the expression of a sequence or gene of interest.
  • the nucleic acid expression vector includes a promoter which is operably linked to the sequences or gene(s) of interest. Other control elements may be present as well.
  • Expression cassettes described herein may be contained within a plasmid construct.
  • the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a bacterial origin of replication e.g., a M13 origin of replication
  • a multiple cloning site e.g., a SV40 or adenovirus origin of replication
  • An “expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest. Such cassettes can be constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target cells.
  • vector vector construct
  • vector vector
  • gene transfer vector gene transfer vector
  • Light-generating is defined as capable of generating light through a chemical reaction or through the absorption of radiation.
  • a “light generating protein” or “light-emitting protein” is a protein capable of generating light in the visible spectrum (between approximately 350 nm and 800 nm). Examples include bioluminescent protiens such as luciferases, e.g., bacterial and firefly luciferases, as well as fluorescent proteins such as green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Light is defined herein, unless stated otherwise, as electromagnetic radiation having a wavelength of between about 300 nm and about 1100 nm.
  • Animal typically refers to a non-human mammal, including, without limitation, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • farm animals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs
  • birds including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • a "transgenic animal” refers to a genetically engineered animal or offspring of genetically engineered animals.
  • a transgenic animal usually contains material from at least one unrelated
  • non-human animals include vertebrates such as rodents, non-human primates, sheep, dogs, cows, amphibians, birds, fish, insects, reptiles, etc.
  • chimeric animal is used to refer to animals in which the heterologous gene is found, or in which the heterologous gene is expressed in some but not all cells of the animal.
  • analyte refers to any compound or substance whose effects (e.g., induction or repression of a specific promoter) can be evaluated using the test animals and methods of the present invention.
  • Such analytes include, but are not limited to, chemical compounds, pharmaceutical compounds, polypeptides, peptides, polynucleotides, and polynucleotide analogs.
  • Many organizations e.g., the National Institutes of Health, pharmaceutical and chemical corporations have large libraries of chemical or biological compounds from natural or synthetic processes, or fermentation broths or extracts. Such compounds/analytes can be employed in the practice of the present invention.
  • positive selection marker refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo 1 ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art. Typically, positive selection markers encode products that can be readily asssayed. Thus, positive selection markers can be used to determine whether a particular DNA construct has been introduced into a cell, organ or tissue.
  • Negative selection marker refers to gene encoding a product which can be used to selectively kill and/or inhibit growth of cells under certain conditions.
  • Non-limiting examples of negative selection inserts include a he ⁇ es simplex virus (HSV)-thymidine kinase (TK) gene. Cells containing an active HSV-TK gene are incapable of growing in the presence of gangcylovir or similar agents. Thus, depending on the substrate, some gene products can act as either positive or negative selection markers.
  • homologous recombination refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of essentially identical nucleotide sequences. It is understood that substantially homologous sequences can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align (see, above).
  • a “knock-out” mutation refers to partial or complete loss of expression of at least a portion the target gene.
  • knock-out mutations include, but are not limited to, gene-replacement by heterologous sequences, gene disruption by heterologous sequences, and deletion of essential elements of the gene (e.g., promoter region, portions of a coding sequence).
  • a "knock-out" mutation is typically identified by the phenotype generated by the mutation.
  • a "single-copy gene” as used herein refers to a gene represented in an organism's genome only by a single copy at a particular chromosomal locus. Accordingly, a diploid organism has two copies of the gene and both copies occur at the same chromosomal location.
  • a "gene” as used in the context of the present invention is a sequence of nucleotides in a genetic nucleic acid (chromosome, plasmid, etc.) with which a genetic function is associated.
  • a gene is a hereditary unit, for example of an organism, comprising a polynucleotide sequence (e.g., a DNA sequence for mammals) that occupies a specific physical location (a "gene locus” or “genetic locus") within the genome of an organism.
  • a gene can encode an expressed product, such as a polypeptide or a polynucleotide (e.g., tRNA).
  • a gene may define a genomic location for a particular event function, such as the binding of proteins and/or nucleic acids (e.g., phage attachment sites), wherein the gene does not encode an expressed product.
  • a gene typically includes coding sequences, such as, polypeptide encoding sequences, and non-coding sequences, such as, promoter sequences, poly-adenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences).
  • non-coding sequences such as, promoter sequences, poly-adenlyation sequences, transcriptional regulatory sequences (e.g., enhancer sequences).
  • Many eucaryotic genes have “exons" (coding sequences) interrupted by "introns” (non-coding sequences).
  • a gene may share sequences with another gene(s) (e.g., overlapping genes).
  • “Isogenic” means two or more organisms or cells that are considered to be genetically identical.
  • “Substantially isogenic” means two or more organisms or cells wherein, at the majority of genetic loci (e.g., greater than 99.000%, preferably more than 99.900%, more preferably greater than 99.990%, even more preferably greater than 99.999%), there exists genetic identity between the organisms or cells being compared.
  • two organisms for example, mice
  • two organisms are considered to be “substantially isogenic” if, for example, inserted transgenes are the primary differences between the genetic make-up of the mice being compared.
  • mice are considered to be substantially isogenic.
  • An example of two strains of substantially isogenic mice are C57BL/6 and C57BL 6-Tyr C2J/+.
  • a "pseudogene" as used herein refers to a type of gene sequence found in the genomes, typically, of eucaryotes, where the sequence closely resembles a known functional gene, but differs in that the pseudogene is non-functional.
  • the pseudogene sequence may contain several stop codons in what would correspond to an open reading frame in the functional gene.
  • Pseudogenes can also have deletions or insertions relative to their corresponding functional gene. If, for example, in a genome there is a functional gene and a related pseudogene, the functional gene is considered to be a single-copy gene (accordingly, the pseudogene is considered to be single-copy as well).
  • non-essential gene refers to a gene whose deletion, disruption, elimination, reduction of gene function, or mutation is non-lethal, and does not obviously adversely affect the organisms' ability to mature and reproduce.
  • non-essential gene with no phenotype refers to a non-essential gene whose deletion, disruption, elimination, reduction of gene function or mutation has no deleterious effect on the organism.
  • a non-essential gene typically one whose function has been eliminated (e.g., by a deletion mutation) and such elimination of function was non-lethal and the organism developed, matured, and was able to reproduce.
  • the "native sequence” or “wild-type sequence” of a gene is the polynucleotide sequence that comprises the genetic locus corresponding to the gene, e.g., all regulatory and open-reading frame coding sequences required for expression of a completely functional gene product as they are present in the wild-type genome of an organism.
  • the native sequence of a gene can include, for example, transcriptional promoter sequences, translation enhancing sequences, introns, exons, and poly-A processing signal sites. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated “polymo ⁇ hisms" or "allelic variations.”
  • replacement sequence is meant a polynucleotide sequence that is substituted for at least a portion of the native or wild-type sequence of a gene.
  • Linear vector or “linearized vector,” as used herein, is a vector having two ends.
  • circular vectors such as plasmids
  • the targeting vectors described herein are linearized such that the ends are not within the targeting sequences.
  • transgenic, non-human mammals are constructed where a single-copy, non-essential gene is replaced by a reporter expression cassette, preferably a gene encoding a light-generating protein, such as a luciferase-encoding gene, operably linked to a promoter.
  • a variety of promoters are useful in the practice of the present invention, for example, promoters derived from genes associated with tumorigenesis or angiogenesis.
  • an exemplary promoter can be one that is associated with proteins induced during tumorigenesis, for instance in the presence of tumor generating compounds or of tumors themselves.
  • expression of the reporter cassette is induced in the animal when, for example, tumors are present, and progression of the tumor can be evaluated by non-invasive imaging methods using the whole animal.
  • Another exemplary promoter is one that is derived from a gene associated with angiogenesis. Because the promoter is linked to a reporter such as luciferase, non- invasive monitoring of the progression of angiogenesis is possible.
  • a reporter such as luciferase
  • the conjugates contain a biocompatible entity and a light-generating moiety.
  • Biocompatible entities include, but are not limited to, small molecules such as cyclic organic molecules; macromolecules such as proteins; microorganisms such as viruses, bacteria, yeast and fungi; eukaryotic cells; all types of pathogens and pathogenic substances; and particles such as beads and liposomes.
  • biocompatible entities may be all or some of the cells that constitute the mammalian subject being imaged, for example, cells carrying the vector constructs of the present invention expressing a reporter expression cassette.
  • Light-emitting capability is conferred on the biocompatible entities by the conjugation of a light-generating moiety.
  • moieties include fluorescent molecules, fluorescent proteins, enzymatic reactions giving off photons and luminescent substances, such as bioluminescent proteins.
  • light emitting capability is typically confered on target cells by having at least one copy of a light- generating protein, e.g., a luciferase, present.
  • luciferase is operably linked to appropriate control elements which can facilitate expression of a polypeptide having luciferase activity.
  • Substrates of luciferase can be endogenous to the cell or applied to the cell or system (e.g., injection into a transgenic mouse, having cells carrying a luciferase construct, of a suitable substrate for the luciferase, for example, luciferin).
  • the conjugation may involve a chemical coupling step, genetic engineering of a fusion protein, or the transformation of a cell, microorganism or animal to express a light-generating protein.
  • the targeting cassettes described herein typically include the following components: (1) a suitable vector backbone; (2) a polynucleotide encoding a light generating protein (3) a promoter operably linked to the light generating protein- encoding gene, wherein the promoter is heterologous to the light generating protein coding sequences; (4) a sequence encoding a positive selection marker; (5) insertion sites flanking the sequence encoding the positive selection marker and the polynucleotide encoding a light generating protein gene, for insertion of sequences which target a single-copy, non-essential chromosomal gene; and, optionally, (6) a sequence encoding a negative selection marker.
  • Exemplary targeting constructs are shown in Figures 3B, 5A and 5B and described in Examples 1-3.
  • Suitable vector backbones generally include an FI origin of replication; a colEl plasmid-derived origin of replication; polyadenylation sequence(s); sequences encoding antibiotic resistance (e.g., ampicillin resistance) and other regulatory or control elements.
  • suitable backbones include: pBluescriptSK (Stratagene, La Jolla, CA); pBluescriptKS (Stratagene, La Jolla, CA) and other commercially available vectors.
  • the light generating protein is luciferase.
  • Luciferase coding sequences useful in the practice of the present invention include, but are not limited to, sequences obtained from lux genes (procaryotic genes encoding a luciferase activity) and luc genes (eucaryotic genes encoding a luciferase activity).
  • lux genes procaryotic genes encoding a luciferase activity
  • luc genes eucaryotic genes encoding a luciferase activity.
  • a variety of luciferase encoding genes have been identified including, but not limited to, the following: B.A. Sherf and K.V. Wood, U.S. Patent No. 5,670,356, issued 23 September 1997; Kazami, J., et al., U.S. Patent No. 5,604,123, issued 18 February 1997; S.
  • Eukaryotic luciferase catalyzes a reaction using luciferin as a luminescent substrate to produce light
  • prokaryotic luciferase catalyzes a reaction using an aldehyde as a luminescent substrate to produce light.
  • Wild-type firefly luciferases typically have an emission maxima at about 550 nm. Numerous variants with differing emission maxima have also been studied. For example, Kajiyama and Nakano (Protein Eng. 4(6):691-693, 1991 ; U.S. Patent No. 5,330,906, issued 19 July 1994) teach five variant firefly luciferases generated by single amino acid changes to the Luciola cruciata luciferase coding sequence. The variants have emission peaks of 558 nm, 595 nm, 607 nm, 609 nm and 612 nm.
  • a yellow-green luciferase with an emission peak of about 540 nm is commerically available from Promega, Madison, WI under the name pGL3.
  • a red luciferase with an emission peak of about 610 nm is described, for example, in Contag et al. (1998) Nat. Med. 4:245-247 and Kajiyama et al. (1991) Prot. Eng. 4:691-693.
  • Positive selection markers include any gene which a product that can be readily asssayed. Examples include, but are not limited to, a hprt gene (Littlefield, J. W., Science 145:709-710 (1964)), a xanthine-guanine phosphoribosyltransferase (gpt) gene, or an adenosine phosphoribosyltransferase (aprt) gene (Sambrook et al., supra), a thymidine kinase gene (i.e "TK”) and especially the TK gene of he ⁇ es simplex virus (Giphart-Gassler, M. et al., Mutat. Res.
  • TK thymidine kinase gene
  • nptll gene a gene which encode enzymes such as dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA), asparagine synthetase (AS), hygromycin B phosphotransferase, or a CAD enzyme (carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase).
  • DHFR dihydrofolate reductase
  • ADA adenosine deaminase
  • AS asparagine synthetase
  • hygromycin B phosphotransferase a CAD enzyme
  • Addition of the appropriate substrate of the positive selection marker can be used to determine if the product of the positive selection marker is expressed, for example cells which do not express the positive selection marker nptll, are killed when exposed to the substrate G418 (Gibco BRL Life Technology, Gaithersburg, MD).
  • the targeting vector typically contains insertion sites for inserting targeting sequences (e.g., sequences that are substantially homologous to the target sequences in the host genome where integration of the targeting vector/expression cassette is desired). These insertion sites are preferably included such that there are two sites, one site on either side of the sequences encoding the positive selection marker, light generating protein (e.g., luciferase) and the promoter. Insertion sites are, for example, restriction endonuclease recognition sites, and can, for example, represent unique restriction sites. In this way, the vector can be digested with the appropriate enzymes and the targeting sequences ligated into the vector.
  • targeting sequences e.g., sequences that are substantially homologous to the target sequences in the host genome where integration of the targeting vector/expression cassette is desired. These insertion sites are preferably included such that there are two sites, one site on either side of the sequences encoding the positive selection marker, light generating protein (e.g., luciferase) and the promote
  • the targeting construct can contain a polynucleotide encoding a negative selection marker.
  • Suitable negative selection markers include, but are not limited to, HSV-tk (see, e.g., Majzoub et al. (1996) New Engl. J. Med. 334:904-907 and U.S. Patent No. 5,464,764), as well as genes encoding various toxins including the diphtheria toxin, the tetanus toxin, the cholera toxin and the pertussis toxin.
  • a further negative selection marker gene is the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene for negative selection in 6-thioguanine.
  • HPRT hypoxanthine-guanine phosphoribosyl transferase
  • the targeting constructs and transgenic animals described herein contain a sequence encoding a light generating protein, e.g., luciferase, gene operably linked to a promoter.
  • the promoter may be from the same species as the transgenic animal (e.g., mouse promoter used in construct to make transgenic mouse) or from a different species (e.g., human promoter used in construct to make transgenic mouse).
  • the promoter can be derived from any gene of interest.
  • the promoter is derived from a gene whose expression is induced during angiogenesis, for example pathogenic angiogenesis like tumor development.
  • the promoter is induced and the animal expresses luciferase, which can then be monitored in vivo.
  • Exemplary promoters for use in the present invention are selected such that they are functional in a cell type and/or animal into which they are being introduced.
  • Exemplary promoters include, but are not limited to, promoters obtained from the following mouse genes: vascular endothelial growth factor (VEGF) (VEGF promoter described in U.S. Patent No. 5,916,763; Shima et al. (1996) J. Bio. Chem. 271 :3877- 3883; sequence available on NCBI under accession number U41383); VEGFR2, also known as Flk-1 , (VEGFR-2 promoter described, for example, in Ronicke et al. (1996) Circ. Res. 79:277-285; Patterson et al. (1995) /.
  • VEGF vascular endothelial growth factor
  • VEGF is a specific mitogen for EC in vitro and a potent angiogenic factor in vivo.
  • VEGF vascular endothelial growth factor
  • VEGF is a major mediator of aberrant EC proliferation and vascular permeability in a variety of human pathologic situation, such as, tumor angiogenesis, diabetic retinopathy and rheumatoid arthritis (Benjamin LE, et al.,1997 PNAS 94: 8761-66; Soker, S., et al.,1998 Cell 92: 735-745).
  • VEGF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels.
  • VEGF plays a pivotal role in promoting tumor growth (Dvorak, H.F., et al., 1991 J Exp Med 174:1275-8). VEGF expression was upregulated by hypoxia (Shweiki, D., et al., 1992 Nature 359:
  • VEGF is also upregulated by overexpression of v-Src oncogene (Mukhopadhyay. D., et al.,1995 Cancer Res. 15: 6161-5), c-SRC (Mukhopadhyay, D., et al., 1995 Nature 375: 577-81), and mutant ras oncogene (Plate, K.H., et al., 1992 Nature 359: 845-8).
  • the tumor suppressor p53 downregulates VEGF expression (Mukhopadhyay. D., et al.,1995 Cancer Res. 15: 6161-5).
  • cytokines and growth factors including PGF and TPA (Grugel, S., et al., 1995 J. Biological Chem. 270: 25915-9), EGF, TGF-b, IL-1 , IL-6 induce VEGF mRNA expression in certain type of cells (Ferrara, N., et al., 1997 Endocr. Rev. 18: 4- 25).
  • Kaposi's sarcoma-associated he ⁇ esvirus encoded a G-protein-coupled receptor, a homolog of IL-8 receptor, can activate JNK/SAPK and p38MAPK and increase VEGF production, thus causing cell transformation and tumorigenicity (Bais, C, et al., Nature 1998 391 :86-9).
  • VEGF overexpression in skin of transgenic mice induces angiogenesis, vascularhype ⁇ ermeability and accelerated tumor development (Larcher, F., et al., Oncogene 1998 17:303-1 1).
  • VEGF-B (cDNA sequences available on databases) is a mitogen for EC and may be involved in angiogenesis in muscle and heart (Olofsson, B., et al., 1996 Proc Natl Acad Sci U S A 93:2576-81). Shown in vitro, binding of VEGF-B to its receptor VEGFR-1 leads to increased expression and activity of urokinase type plasminogen activator and plasminogen activator inhibitor, suggesting a role for VEGF-B in the regulation of extracellular matrix degradation, cell adhesion, and migration (Olofsson, B., et al., 1998 Proc Natl Acad Sci U S A 95:11709-14).
  • VEGF-C may regulate angiogenesis of lymphatic vasculature, as suggested by the pattern of VEGF-C expression in mouse embryos (Kukk, E., et al., 1996 Development 122: 3829-37). Although VEGF-C is also a ligand for VEGFR-2, the functional significance of this potential interaction is unknown.
  • VEGF-C Overexpression of VEGF-C in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement, suggesting the major function of VEGF-C is through VEGFR-3 rather than VEGFR-2 (Jeltsch M, et al., 1997 Science 276:1423-5). Shown by the CAM assay, VEGF and VEGF-C are specific angiogenic and lymphangiogenic growth factors, respectively (Oh, S.J., et al., (1997) Devel. Biol. 188: 96-109). VEGF-C overexpression in the skin of transgenic mice resulted in lymphatic, but not vascular, endothelial proliferation and vessel enlargement (Jeltsch M, et al., 1997 Science 276: 1423-5).
  • VEGF-D (cDNA sequences available on databases) is a mitogen for EC. Given that VEGF-D can also activate VEGFR-3. it is possible that VEGF-D could be involved in the regulation of growth and or differentiation of lymphatic endothelium (Achen, M.G., et al., 1998 Proc Natl Acad Sci U S A 95: 548-53). VEGF-D is induced by transcription factor c-Fos in mouse (Orlandini, M., 1996 PNAS 93: 11675-80).
  • VEGFR-1 signaling pathway may regulate normal endothelial cell-cell or cell matrix interactions during vascular development, as suggested by the knockout study (Fong, G.H., et al., 1995 Nature 376: 65-69).
  • VEGFR-1 has a higher affinity to VEGF than VEGFR-2, it does not transduce the mitogenic signals of VEGF in ECs (Soker, S., et al.,1998 Cell 92: 735-745).
  • VEGFR-2 see, e.g., Ronicke et al., Patterson et al., Kappel et al.
  • VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of lymphatic vessels, as shown by the knockout study (Dumont, D.J., et al., 1998 Science 282: 946- 949).
  • Neuropillin-1 is a receptor for VEGF165.
  • Ang2 is expressed only at predominant vascular remodeling sites, such as ovary, placenta, uterus (Maisonpierre, P.C., et al., 1997 Science 277: 55-60).
  • vascular remodeling sites such as ovary, placenta, uterus
  • Ang2 is found to be expressed in endothelial cells of small blood vessel and capilaries while Angl is expressed in glioblastoma tumor cells (Stratmann, A., 1998 Am J Pathol 153: 1459-66).
  • Ang2 is up-regulated in bovine microvascular endothelial by VEGF, bFGF, cyrokines, hypoxia (Mandriota, S.J., 1998 Circ Res 83: 852-9).
  • Ang2 transgenic overexpression disrupts angiogenesis, and is embryonic lethal (Maisonpierre, P.C., et al., 1997 Science 277: 55-60).
  • Angl is widely expressed, less aboundant in heart and liver (Maisonpierre, P.C., et al., 1997 Science 277: 55-60).
  • Angl is expressed in mesenchymal cells and may up-regulate the expression of Tie2 in the endothelial cells (Suri,C, et al., 1996 Cell 87: 1171-1 180). Angl overexpression in the skin of transgenic mice produces larger, more numerous, and more highly branched vessels (Suri, C, et al., Science 1998 282:468-71 ). Tie2 (see, e.g., Fadel et al.; Schlaeger et al. (1995), and Schlager et al.
  • a Tie2 activating mutation causes vascular dysmorphorgenesis (Vikkula M, et al., 1996 Cell 87: 1181 -1 190). Tie2 mutant overexpression in transgenic mice is embryonic lethal (Dumont, D.J., et al., supra).
  • Other promoters useful in the practice of the present invention include, by way of example, promoters derived from the sequences encoding the following polypeptide products: PTEN (dual specificity phosphatase); BAI (brain-specific angiogenesis inhibitor); KAI1 (KANGAI 1); catenin beta-1 (cadherin-associated protein, beta); COX2 5 (PTGS2 cyclooxygenase 2, a.k.a.
  • prostaglandin-endoperoxide synthase 2 MMP2 (72 kDa Type IV-A collagenase); MMP9 (92 kDa type IV-B collagenase); TIMP2 (tissue inhibitor of metalloproteinase 2); and TIMP3 (tissue inhibitor of metalloproteinase 3).
  • PTEN is a tumor suppressor gene and encodes a protein of 403 amino acids.
  • BAH protein is predicted to be 1 ,584 amino acids in length and includes an extracellular domain, an intracellular domain and a 7-span transmembrane region similar to that of the secretin receptor. (Nishimori et al. (1997) Oncogene 15:245-2150).
  • the 5 extracellular region of BAH has a single Arg-Gly-Asp (RGD) motif recognized by integrins and also has five sequences corresponding to the thrombospondin type I (accession number 188060) repeats that can inhibit angiogenesis includes by basic fibroblast growth factor (bFGF, accession number 134920). Shiratsuchi et al. (1997) Cytogenet. Cell Genet. 79:103-108, cloned 2 other brain-specific angiogenesis inhibiting
  • KAIl encodes a 267 amino acid protein which is a member of the leukocyte surface glyoprotein family.
  • the protein has 4 hydrophobobic transmembrane domains and 1 large extracellular hydrophilic domain with three potential N-glycosylation sites. (Dong et al. (1995) Science 268:884-886). Molecular analysis of KAIl is described, for example, in Dong et al. (1997) Genomics 41 :25-32.
  • KAIl is a tumor metastasis suppressor gene that is capable of inhibiting the metastatic process in experimental animals. Expression of KAIl is downregulated during tumor progression of prostate, breast, lung, bladder and pancreatic cancers in humans, apparently at the transcriptional or postranscriptional level. Mashimo et al. (1998) PNAS USA 95:1 1307-1131 1, found that the tumor suppressor gene p53 can directly inactivate the KAIl gene by interacting with the region 5' to the coding sequence, suggesting a direct relationship between p53 and KAIl.
  • Catenin beta-1 is an adherens junction (AJ) protein, which are critical for establishing and maintaining epithelial cell layers, for instance during embryogenesis, wound healing and tumor cell metastasis.
  • AJ adherens junction
  • COX2 is generally considered to be a mediator of inflammation and overexpression of COX2 in rat epithelial cells results in elevated levels of E-cadherin and Bcl2.
  • overexpression of COX2 in rat epithelial cells results in elevated levels of E-cadherin and Bcl2.
  • cells that overexpress COX2 produce prostaglandins, proangiogenic factors and stimulate both endothelial migration and tube formation.
  • APC knock-out mice have demonstrated that animals homozygous for a disrupted COX2 locus develop significantly more adenomatous polyps.
  • MMP2 is a metalloproteinase that specifically cleaves type IV collagen.
  • MMP9 is a collagenase secreted from normal skin fibroblasts. MMP9 null mice exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. (Vu et al. (1998) Cell 93:411-422). TIMP2 is a collagenase and appears to play a major role in modulating the activity of interstitial collagenase and a number of connective tissue metalloendoproteases. (Stetler-Stevenson et al. (1989) 7. Biol. Chem. 264:17372-17378). Unlike TIMP1 and TIMP3, TIMP2 is not upregulated by TPA or TGF-beta. (Hammani et al. (1996) J. Biol. Chem.
  • TIMP3 (Wilde et al. (1994) DNA Cell Biol. 13:71 1-718) is localized in the extracellular matrix in both its glycosylated and unglycosylated forms. Studies of mutant TIMP3 proteins have demonstrated that C-terminal trunctions do not bind to the extracellular matrix. (Langton et al. (1998) J. Biol. Chem. 273:16778-16781 ).
  • promoter sequences can be easily derived and isolated from known polypeptide sequences or from cDNA or genomic sequences, using method known in the art in view of the teachings herein.
  • An exemplary method of isolating promoter sequences using cDNA is via a GenomeWalker® kit, commercially available from Clontech (Palo Alto, CA), and described on page 27 of the 1997-1998 Clontech catalog.
  • Targeting Sequences Non-Essential Genes Central to the present invention is the fact that the targeting constructs contain
  • targeting sequences flankking, for example, the light generating protein-encoding sequence and promoter
  • These targeting sequences in the construct act via homologous recombination to replace at least a portion of the non-essential gene in the genome with the light-generating protein-encoding (e.g., luciferase-encoding) sequence operably linked to a promoter.
  • the light-generating protein-encoding e.g., luciferase-encoding
  • Non-limiting examples of targeting sequences for use in generating transgenic mice include sequences obtained from or derived from vitronectin, Fos B and galactin 3.
  • a search of Mouse Knockout & Mutation Database can be used to identify genes that have been knocked-out in mice where the generated knockout mice displayed no obvious defects.
  • the chromosomal locus for all these genes can be used to target promoter-(light generating protein, e.g., luciferase) transgenes similar to what is described in Example 2.
  • Single-copy, non-essential mouse genes identified in this manner include, but are not limited to, the following: Moesin (Msn), Doi Y., et al., J Biol Chem 1999, 274:2315-2321 ; Plasminogen activator inhibitor, type II (Planh2) and Planhl , Dougherty K.M., Proc Natl Acad Sci USA 1999, 96:686-691 ; Protein tyrosine phosphatase, receptor type, B (Ptprb), Elchebly et al. (1999) Science 283:1544-1548; Presenilin 1 (Psenl), Guo Q, et al.
  • Hemochromatosis Hfe
  • Zhou XY et al. Proc Natl Acad Sci USA 1998, 95:2492-2497
  • Alpha tropomyosin Tpml
  • Blanchard EM et al. Circ Res 1997, 81: 1005-1010
  • tRNA phosphoserine Trsp
  • Bosl MR et al. Proc Natl Acad Sci U S A 1997, 94:5531-5534
  • Angiotensin receptor lb Agtrlb
  • Chen X et al.
  • TNF-R-1 Tumor necrosis factor receptor 1
  • Pfeffer K Tumor necrosis factor receptor 1
  • Lgalsl Tumor necrosis factor receptor 1
  • Pfeffer K Tumor necrosis factor receptor 1
  • Lectin galactose binding, soluble 1 (Lgalsl), Poirier F, Robertson EJ. 5 Development 1993, 119:1229-1236
  • Synapsin I Synl
  • Rosahl TW et al. Cell 1993, 75:661-670
  • Tumor necrosis factor receptor 1 Tumor necrosis factor receptor 1
  • Rothe J et al. Nature 1993, 364:798-802
  • Beta-2 microglobulin (B2m) Correa I, et al.
  • Some preferred single-copy, non-essential genes with no phenotypes of the present invention include, but are not limited to, the following: Moesin (Msn), Doi Y., et al., J Biol Chem 1999, 274:2315-2321; Plasminogen activator inhibitor, type II (Planh2) and Planhl , Dougherty K.M., Proc Natl Acad Sci USA 1999, 96:686-691 ; Nuclear receptor coactivator 1 (Ncoal), Qi C, et al. (1999) Proc Natl Acad Sci USA 96: 1585-
  • the targeting cassettes described herein can be constructed utilizing methodologies known in the art of molecular biology (see, for example, Ausubel or
  • the targeting constructs are assembled by inserting, into a suitable vector backbone, polynuclotides encoding a reporter, such as a light-generating protein, e.g., a luciferase gene, operably linked to a promoter of interest; a sequence encoding a positive selection marker; and, optionally a sequence encoding a negative selection marker.
  • a reporter such as a light-generating protein, e.g., a luciferase gene
  • the targeting cassette contains insertion sites such that sequences targeting a single-copy, non-essential gene can be readily inserted to flank the sequence encoding positive selection marker and luciferase-encoding sequence.
  • PCR A preferred method of obtaining polynucleotides, suitable regulatory sequences (e.g., promoters) is PCR.
  • suitable regulatory sequences e.g., promoters
  • PCR conditions for each application reaction may be empirically determined.
  • a number of parameters influence the success of a reaction. Among these parameters are annealing temperature and time, extension time, Mg2+ and ATP concentration, pH, and the relative concentration of primers, templates and deoxyribonucleotides. Exemplary primers are described below in the Examples.
  • the resulting fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.
  • PCR can be used to amplify fragments from genomic libraries.
  • Many genomic libraries are commercially available.
  • libraries can be produced by any method known in the art.
  • the organism(s) from which the DNA is has no discernible disease or phenotypic effects.
  • This isolated DNA may be obtained from any cell source or body fluid (e.g., ES cells, liver, kidney, blood cells, buccal cells, cerviovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy, urine, blood, cerebrospinal fluid (CSF), and tissue exudates at the site of infection or inflammation).
  • DNA is extracted from the cells or body fluid using known methods of cell lysis and DNA purification.
  • the purified DNA is then introduced into a suitable expression system, for example a lambda phage.
  • a suitable expression system for example a lambda phage.
  • Another method for obtaining polynucleotides, for example, short, random nucleotide sequences, is by enzymatic digestion. As described below in the Examples, short DNA sequences generated by digestion of DNA from vectors carrying genes encoding luciferase (yellow green or red).
  • Polynucleotides are inserted into vector genomes using methods known in the art.
  • insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary or blunt ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Other means are known and, in view of the teachings herein, can be used.
  • the final constructs can be used immediately (e.g., for introduction into ES cells), or stored frozen (e.g., at -20°C) until use.
  • the constructs are linearized prior to use, for example by digestion with suitable restriction endonucleases.
  • the targeting constructs containing the light generating protein coding sequences are introduced into a pluripotent cell (e.g., ES cell, Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N. Y. ( 1989), pp. 39-44).
  • a pluripotent cell e.g., ES cell, Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N. Y. ( 1989), pp. 39-44.
  • Suitable ES cells may be derived or isolated from any species or from any strain of a particular species.
  • the pluripotent cells are typically derived from the same species as the intended reciepient.
  • ES cells may be obtained from commercial sources, from International Depositories (e.g., the ATCC) or, alternatively, may be obtained as described in Robertson, E. J., supra.
  • ATCC International Depositories
  • Examples of clonally-derived ES cells lines include 129/SVJ ES cells, RW-4 and C57BL/6 ES cells (Genome Systems, Inc.).
  • ES cells are cultured under suitable conditions, for example, as described in Ausubel et al., section 9.16, supra.
  • ES cells are cultured on stomal cells (such as STO cells (especially SNC4 STO cells) and/or primary embryonic fibroblast cells) as described by E. J. Robertson, supra, pp 71-112.
  • Culture media preferably includes leukocyte inhibitory factor ("lif") (Gough, N. M. et al., Reprod. Fertil. Dev. 1 :281-288 (1989); Yamamori, Y. et al., Science 246:1412-1416 (1989), which appears to help keep the ES cells from differentiating in culture.
  • Stomal cells transformed with the gene encoding lif can also be used.
  • the targeting constructs are introduced into the ES cells by any method which will permit the introduced molecule to undergo recombination at its regions of homology, for example, micro-injection, calcium phosphate transformation, or electroporation (Toneguzzo, F. et al., Nucleic Acids Res. 16:5515-5532 (1988); Quillet, A. et al., J. Immunol. 141 : 17-20 (1988); Machy, P. et al., Proc. Natl. Acad, Sci. (U.S.A.) 85:8027-8031 (1988)).
  • the construct to be inserted into the ES cell must first be in the linear form.
  • the knockout construct has been inserted into a vector as described above, linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence. If the ES cells are to be electroporated to insert the construct, the ES cells and construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then cultured under conventional conditions, as are known in the art, and screened for the presence of the construct.
  • the construct contains both positive and negative selection markers.
  • methods which rely on expression of the selection marker are used, for example, by adding the appropriate substrate to select only those cells which express the product of the positive selection marker or to eliminate those cells expressing the negative selection marker.
  • the positive selection marker encodes neomycin resistance
  • G418 is added to the transformed ES cell culture media at increasing dosages.
  • a suitable substrate e.g., gancyclovir if the negative selection marker encodes HSV-TK
  • the presence of the positive and/or negative selection markers in a recipient cell can also be determined by others methods, for example, hybridization, detection of radiolabelled nucleotides, PCR and the like.
  • cells having integrated targeting constructs are first selected by adding the appropriate substrate for the positive and/or negative selection markers. Cells that survive the selection process are then screened by other methods, such as PCR or Southern blotting, for the presence of integrated sequences.
  • the cells can be inserted into an embryo, preferably a blastocyst.
  • the blastocyts are obtained by perfusing the uterus of pregnant females.
  • the blastocyts are obtained from, for example, the FVB/N strain of mice and the ES cells are obtained from, for example, the C57BL/6 strain of mice. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al., (1992) Biotechnology, 10:534-539. Insertion into the embryo may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection.
  • ES cells For microinjection, about 10-30 ES cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the construct into the developing embryo.
  • the suitable stage of development for the embryo used for insertion of ES cells is species dependent, in mice it is about 3.5 days.
  • blastocysts While any embryo of the right stage of development is suitable for use, it is preferred that blastocysts are used.
  • preferred blastocysts are male and, furthermore, preferably have genes encoding a coat color that is different from that encoded by the genes ES cells.
  • the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was inco ⁇ orated into the developing embryo).
  • the blastocyst selected will carry genes for white or brown fur.
  • the blastocyst is typically implanted into the uterus of a pseudopregnant foster mother for gestation.
  • Pseudopregnant females are prepared by mating with vasectomized males of the same species and successful implantation usually must occur within about 2-3 days of mating.
  • Offspring are screened initially for mosaic coat color where the coat color selection strategy has been employed. Southern blots and/or PCR may also be used to determine the presence of the sequences of interest.
  • Mosaic (chimeric) offspring are then bred to each other to generate homozygous animals.
  • Homozygotes and heterozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.
  • Northern blots can be used to probe the mRNA to identify the presence or absence of transcripts encoding either the replaced gene, the light generating protein coding sequence (e.g., luciferase gene), or both.
  • Western blots can be used to assess the level of expression of the luciferase protein with an antibody against the luciferase gene product.
  • in situ analysis such as fixing the cells and labeling with antibody
  • FACS fluorescence activated cell sorting
  • the animals are from the C57BL/6 mouse strain.
  • This strain develops a variety of tumors and has been used to develop a number of tumor cells lines, for example, B16 melanoma cells (including, B 16F10, B 16D5, and B 16F1), Lewis lung carcinoma cells (including, LLC, LLC-h59), T241 mouse fibrosarcoma cells, RM-1 and pTC2 mouse prostate cancer cells, and MCA207 mouse sarcoma cells.
  • B16 melanoma cells including, B 16F10, B 16D5, and B 16F1
  • Lewis lung carcinoma cells including, LLC, LLC-h59
  • T241 mouse fibrosarcoma cells RM-1 and pTC2 mouse prostate cancer cells
  • MCA207 mouse sarcoma cells MCA207 mouse sarcoma cells.
  • the generated targeted transgenic mice in the Examples are in C57BL/6 genetic background and these animals are suitable for injection or implantation of such tumor cells, as well as other tumor cells described in literature that are immunocompatent for C57BL/6 mice.
  • the transgenic animals can then be used, for example, to monitor, in vivo, tumor progression (e.g., growth) and the efficacy of therapies on tumor regression.
  • tumor progression e.g., growth
  • efficacy of therapies on tumor regression e.g., cancer
  • the transgenic animal is tumor-susceptible, it is monitored for expression of a reporter, e.g., luciferase, which is indicative of tumorigenesis and/or angiogenesis.
  • a reporter e.g., luciferase
  • Such imaging typically uses at least one photo detector device element, for example, a charge -coupled device (CCD) camera.
  • CCD charge -coupled device
  • the transgenic animals described herein can also be used to determine the effect of an analyte (e.g., therapy), for example on tumor progression where the promoter induces light generating protein (e.g., luciferase) expression when a tumor develops.
  • Methods of administration of the analyte include, but are not limited to, injection (subcutaneously, epidermally, intradermally), intramucosal (such as nasal, rectal and vaginal), intraperitoneal, intravenous, oral or intramuscular.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the analyte of interest can be administered over a range of concentration to determine a dose/response curve.
  • the analyte may be administered to a series of test animals or to a single test animal (given that response to the analyte can be cleared from the transgenic animal).
  • the following examples are intended only to illustrate the present invention and should in no way be construed as limiting the subject invention.
  • NeoF SEQ ID NO: 3: ACCTGCAGCCAATATGGGATCGGCCATTGAAC
  • NeoR SEQ ID NO4: GGATCCGCGGCCGCCCCCAGCTGGTTCTTTCCGCCTC
  • TKR SEQ ID NO:6: GAGCTCCCGTAGTCAGGTTTAGTTCGTCCG
  • a synthetic linker F5R5 was made after annealing of two primers (forward primer, F5R51 , SEQ ID NO: 7: GTACATTTAAATCCTGCAGG; reverse primer, F5R52, SEQ ID NO:8: AGCTCCTGCAGGATTTAAAT). This linker was inserted between Asp718I and Hindlll sites of pTK and the new construct was designated pTK5.
  • a second synthetic linker F3R3 was made by annealing of two primers (forward primer, F3R31 , SEQ ID NO:9:
  • Luciferase pTK-LucYG and pTK-LucR The yellow green luciferase gene was isolated from pGL3 vector (Promega) as a Hindlll-Sall fragment and was cloned into pGK53 that was linearized with the same enzymes. The new construct was designated pTK-LucYG (8931 bp), shown in Figure 2.
  • the red luciferase gene was isolated from pGL3-red vector (Dr. Christopher
  • vitronectin targeting vector The targeting construct pTKLR-Vn was generated by inserting vitronectin (VN) DNA sequences into pTK-LucR vector.
  • VN Vitronectin
  • FIG. 3 shows the restriction map of pTKLR-Vn vector.
  • the polylinker between the neomycin gene and red luciferase gene is used to insert the VEGF promoter or other promoters of interests.
  • the predicted homologous recombination between pTKLR-Vn and vitronectin gene is illustrated in Figure 3A. Upon insertion of the VEGF-LucR transgene cassette, the endogenous vitronectin gene is destroyed.
  • Figure 3B shows the genomic DNA sequence of VN.
  • the targeting construct pTKLG-Fos was generated by inserting FosB DNA sequences into pTK-LucYG vector.
  • FosB is one of the members of the Fos family. It plays a functional role in transcriptional regulation. It has been shown that FosB mice are born at a normal frequency, are fertile and present no obvious phenotypic or histologic abnormalities (Gruda et al (1996) Oncogene 12:2177-2185). A 28.8 kb genomic region that contains mouse FosB DNA sequence was obtained from GenBank database (Accession number AF093624).
  • a 1.58 kb 3'end FosB fragment was amplified (forward primer, FosB2F, SEQ ID NO: 17: AACGCGTCGACGGATGGGATTGACCCCCAGCCCTC; reverse primer, FosB2R, SEQ ID NO: 18: TTGGCGCGCCCCTTGCCTCCACCTCTCAAATGC) using mouse C57BL/6 genomic DNA as template.
  • This fragment was digested with Sail and Ascl and cloned into pTK-LucYG vector that was linearized with Sail and Ascl. This construct was designated as pTKLG-Fos ( Figure 4).
  • the polylinker between the neomycin gene and red luciferase gene is used to insert the VEGFR2 promoter (Example 3, Figure 5A), Tie2 promoter (Example 3, Figure 5B), as well as, other promoters of interests.
  • the predicted homologous recombination between the targeting vector bearing the VEGFR2 promoter ( Figure 5A) or the Tie2 promoter ( Figure 5B) and FosB gene is also illustrated.
  • the VEGFR2-LucYG transgene cassette and Tie2-LucYG transgene cassette is inserted downstream of FosB gene translational stop signal.
  • the targeted transgenic mice should still have a functional FosB gene while expressing the transgenes.
  • Figure 4B showns the DNA sequence of FosB.
  • pTKLR-Vn/VEGF Mouse VEGF genomic DNA sequence of 2240 bp that contains a partial VEGF promoter region was obtained from GenBank (accession number: U41383). Accordingly, primers were designed to amplify a 0.69 kb (VF1- VR1A; Table 1) and a 0.98 kb fragment (VF2-VR2; Table 1). It was confirmed that each pair of primers can amplify the predicted product using mouse 129SvJ genomic DNA as template.
  • VEGFR2 promoter region was published previously (Ronicke, et al., (1996) Cir. Res.
  • primers were designed to amplify a 0.45 kb (KFl-KRl A;
  • each pair of primers can amplify the predicted product using mouse 129SvJ genomic DNA as template.
  • DNA sequences for these primers are shown in Table 1 above and PCR amplification conditions are shown in Figure 6.
  • These primers were used for PCR screening of mouse 129/SvJ genomic DNA BAC library. From the screening, a BAC clone that contained a large genomic DNA fragment was obatined.
  • VEGFR2 BAC clone a 4.5 kb Hindlll-Xbal fragment that covers the VEGFR2 promoter region was subcloned from the VEGFR2 BAC clone into the pBluescriptSK vector (Stratagene, La Jolla, CA) that was linearized with Hindlll and Xbal.
  • a BAC clone containing a large genomic DNA fragment of the Tie2 promoter region was obtained. Based on the published Tie2 genomic DNA restriction map (see, Dumont et al., supra), a 10.5 kb Asp718-EcoRV fragment containing the Tie2 promoter region was subcloned ifrom the Tie2 BAC clone into the pSK vector that was linearized with Asp718 and EcoRV. The Tie2 promoter sequences of about 6.8 kb, spanning from the Asp718 site to the ATG translational start codon is subcloned into the polylinker of pTKLG-Fos vector to construct the Tie2-LucYG targeting vector ( Figure 5B).
  • Figure 7 depicts a generalized description of generation of transgenic mice using the targeted transgenic vectors described in Example 3. Details regarding embryonic stem (ES) cell culture, transfection, blastocyst injection and implantation to a pseudopregnant foster are described, for example, in Hogan et al (1994) "Manipulating the Mouse Embryo, A Laboratory Manual. Second Edition", Cold Spring Harbour Laboratory Press.
  • ES embryonic stem
  • the targeted transgenic construct are transfected into C57BL/6 embryonic stem (ES) cells.
  • ES C57BL/6 embryonic stem
  • the antibiotic G418 is used to select for cells in which the DNA construct containing the Neo gene is integrated, either randomly or by homologous recombination.
  • the nucleoside analog gancyclovir is converted by TK to a cytotoxic derivative. DNA that has integrated by homologous recombination lose the TK gene and are resistant to the drug, whereas cells that have inco ⁇ orated the DNA randomly are likely to retain the TK gene.
  • cells containing random integrations into a chromosomal location that allows the expression of the TK gene are killed.
  • the G418 and gancyclovir resistant clones are then be screened by PCR and Southern blot analysis and those that have homologous DNA recombination is used for FVB N blastocyst injection (Genome System, Inc.). Between 4-16 blastocysts are transferred to the uterus of a pseudopregnant foster mother. The pups are typically born 17 days after the transfer. Either random bred mice or FI hybrid mice make suitable recipients. Females of certain random-bred stocks (e.g., CDl mice, from Charles River Laboratories) have very large ampullae, which makes oviduct transfer easier. These mice also generally make good mothers.
  • FI hybrid females e.g., B6 x CBA FI
  • FI hybrid females e.g., B6 x CBA FI
  • their ampullae are smaller, make exceptionally good mothers,rearing litters as small as two pups. See, for example, Hogan et al. (1994), supra.
  • pTKLG-Fos/VEGFR2 Analysis of homologous DNA recombination between pTKLG-Fos/VEGFR2 targeting vector and the FosB gene is carried out using Southern blot analysis as shown in Figure 8.
  • Genomic DNA prepared from G418 resistant ES cells is digested with PvuII and probed with probe A to confirm the 5'end DNA recombination.
  • PvuII digestion of DNA bearing homologous recombination reveals two separate bands of 8.2 and 4.0 kb, whereas digestion of DNA from homologous recombination negative clones reveals only the 8.2 kb band.
  • the 3'end of DNA recombination is tested by hybridizing Notl digested DNA with probe B. Notl digestion of DNA bearing homologous recombination will reveal two separate bands of >8.2kb and 5.0 kb, whereas digestion of DNA from homologous recombination negative clones will only reveal the >8.2 kb band.
  • pTKLG-Fos/Tie2 Analysis of homologous DNA recombination between pTKLG-Fos/Tie2 targeting vector and the FosB gene is analyzed by Southern blot in a similar manner as described above for pTKLG-Fos VEGFR2. Once homologous DNA recombination is confirmed, positive clones are selected for FVB/N blastocyst injection.
  • PTKLR-Vn/VEGF Analysis of homologous DNA recombination between pTKLR-Vn/VEGF targeting vector and the vitronectin gene is analyzed by PCR. DNA primers designed according to the predicted homologous recombination, are listed in Table 2.
  • Primers F51-R51 and F52-R52 amplify a 1799 bp and a 1841 bp DNA fragment respectively from the 5'end of the transgene that is integrated into the vitronectin site through homologous DNA recombination
  • primers F31-R31 and F32-R32 amplify a 3549 bp and a 3428 bp DNA fragment respectively from the 3'end of the transgene that is integrated into the vitronectin site through homologous DNA recombination.
  • Clones that allow successful amplification of both the 5'end and 3'end of the integrated transgene are selected for FVB/N blastocyst injection.
  • the pups developed from injected blastocysts contain chimeras, as can be identified by their agouti coat color when an ES cell derived from a mouse having a dark coat color (e.g., C57BL/6) is injected into the blastocyst of a light coat color animal (e.g., FVB/N, genotype B/B).
  • DNA analysis e.g., Southern blotting, PCR
  • these animals may be obtained commerically, for example from The Jackson Laboratory, Bar Harbor, MN. D. Generating targeted transgenic C57BL/6 mice with white coat color
  • the targeted mice are used to monitor gene expression through the measurement of luciferase mediated light emission from the mice.
  • the targeted mouse has a light coat color (e.g., white coat color), because the black colored coat (an example of a dark coat color) of C57BL 6 mice can absorb light emitted from the body and may interfere the sensitivity of the bioluminescence assay.
  • An inbred mouse strain C57BL/6-Tyr C2j/+ strain (Jackson Laboratory, Bar Harbor, MN) is available for this pu ⁇ ose.
  • mice have white color coat, yet they still have the same genetic background as C57BL/6 mice except that the gene responsible for the black coat color is mutated.
  • C57BL/6-Tyr C2j/+ ES cells are not currently available. Therefore, the designed breeding program illustrated in Figure 9 is aimed to generate mice that are homozygous for the target transgene and have white coat color.
  • C57BL/6 ES cells are prepared as described above and introduced into a suitable blastocyst (e.g., from the FVB/N strain of mice). The blastocysts are implanted into a foster mother. Chimeric mice are shown in Figure 9 as white animals with black and green patches.
  • Chimeric animals are bred with C57BL 6-Tyr C2j/+ mice to create FI hybrids. Subsequent breeding of the FI hybrids generates several type of mice, including the one that is homozygous for the target transgene and has a white coat color (shown in Figure 9 as b/b; L/L), which is used for in vivo gene regulation monitoring.
  • a C57BL 6 mouse and a C57BL/6-Tyr C2j/+ mouse are considered to be substantially isogenic. Accordingly, the method of the present invention exemplified in Figure 9 provides a means for generating breeding groups of substantially isogenic mice in a selected genetic background carrying at least one transgene of interest.
  • PTKLR-Vn carries a red luciferase gene and is targeted into vitronectin locus.
  • PTKLG-Fos carries a yellow- green luciferase gene and is targeted into FosB locus.
  • a number of promoters, including VEGF promoter, VEGFR2 promoter, and Tie2 promoter are cloned into these vectors, as described above.
  • VEGF mice carry VEGF promoter-red luciferase transgene (VEGF-LucR) integrated into vitronectin locus.
  • VEGFR2 mice carry VEGFR2 promoter-yellow-green luciferase (VEGFR2-LucYG) transgene integrated into FosB locus.
  • Tie2 mice carry Tie2 promoter- yellow-green luciferase (Tie2-LucYG) transgene integrated into FosB locus.
  • dual luciferase targeted transgenic mice are produced, carrying both of the VEGF-LucR and the VEGFR2-LucYG transgenes.
  • the degradation of luciferin by yellow-green luciferase and red luciferase generates lights that emit at 540 nM and 610 nM respectively. These wavelengths of light are measured individually using a photo-counting camera (intensified CCD). Therefore, both VEGF expression and VEGFR2 expression, for example, can then be monitored in the same mouse at the same time.

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Abstract

La présente invention concerne des constructions de ciblage et des procédés pour les utiliser afin de créer des animaux transgéniques. Selon ces procédés, au moins un gène à copie unique, non essentiel, est remplacé par une cassette d'expression de rapporteur, par exemple un gène de luciférase, lié de façon opérationnelle à un promoteur hétérologue du gène à copie unique, non essentiel. La présente invention concerne ainsi de nouveaux procédés et de nouvelles constructions de vecteurs qui sont utilisés pour générer des animaux transgéniques. La présente invention concerne également des procédés pour utiliser lesdits animaux.
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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002093143A2 (fr) 2001-05-17 2002-11-21 Xenogen Corporation Procede et appareil pour determiner la profondeur, la brillance et la dimension d'une cible dans une zone du corps humain
US6610503B1 (en) 1999-03-17 2003-08-26 Xenogen Corporation Animal models for predicting sepsis mortality
US6647179B2 (en) 1999-12-21 2003-11-11 Agilent Technologies, Inc. Process and device for making gratings in optical fibres
US6649143B1 (en) 1994-07-01 2003-11-18 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6737245B1 (en) 1999-09-08 2004-05-18 Xenogen Corporation Luciferase expression cassettes and methods of use
EP1373473A4 (fr) * 2001-12-04 2005-02-16 Genome Biosciences Llc Procedes et vecteurs de ciblage genetique
US6867348B1 (en) 1999-12-16 2005-03-15 Xenogen Corporation Methods and compositions for screening for angiogenesis modulating compounds
US7056728B2 (en) 2000-07-06 2006-06-06 Xenogen Corporation Compositions and methods for use thereof in modifying the genomes of microorganisms
WO2007026173A1 (fr) * 2005-09-02 2007-03-08 Birkeland Innovasjon As Modele d'animal transgenique pour identification de modulateurs de la proteine kinase
EP1808441A1 (fr) * 2006-01-11 2007-07-18 AXXAM S.p.A. Cellules souches luminescentes, mammifères transgéniques et leur utilisation
WO2007080621A1 (fr) * 2006-01-11 2007-07-19 Axxam S.P.A. Animaux transgéniques non humains luminescents, leur progéniture, leurs dérivés cellulaires et leurs applications
US7298415B2 (en) 2001-07-13 2007-11-20 Xenogen Corporation Structured light imaging apparatus
US7449615B2 (en) 1998-12-17 2008-11-11 Xenogen Corporation Non-invasive evaluation of physiological response in a transgenic mouse
US7555332B2 (en) 2002-07-16 2009-06-30 Xenogen Corporation Fluorescent light tomography
US7603167B2 (en) 2002-07-16 2009-10-13 Xenogen Corporation Method and apparatus for 3-D imaging of internal light sources
US8044996B2 (en) 2005-05-11 2011-10-25 Xenogen Corporation Surface construction using combined photographic and structured light information
US8545814B2 (en) 1994-07-01 2013-10-01 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US10775308B2 (en) 2006-08-24 2020-09-15 Xenogen Corporation Apparatus and methods for determining optical tissue properties
US11730370B2 (en) 2006-08-24 2023-08-22 Xenogen Corporation Spectral unmixing for in-vivo imaging

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023633A1 (fr) * 1996-11-27 1998-06-04 Cornell Research Foundation, Inc. Identification de genes de mammiferes
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO1998030715A1 (fr) * 1997-01-07 1998-07-16 California Institute Of Technology Detecteur optique de signalisation cellulaire
WO1998036081A2 (fr) * 1997-02-13 1998-08-20 Memorial Sloan-Kettering Cancer Center Molecules hybrides pour la detection optique des modifications des micro-environnements cellulaires

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998023633A1 (fr) * 1996-11-27 1998-06-04 Cornell Research Foundation, Inc. Identification de genes de mammiferes
WO1998028971A2 (fr) * 1997-01-03 1998-07-09 University Technology Corporation Toxicite de la beta-amyloide
WO1998030715A1 (fr) * 1997-01-07 1998-07-16 California Institute Of Technology Detecteur optique de signalisation cellulaire
WO1998036081A2 (fr) * 1997-02-13 1998-08-20 Memorial Sloan-Kettering Cancer Center Molecules hybrides pour la detection optique des modifications des micro-environnements cellulaires

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CONTAG CH, ET AL.: "Visualizing gene expression in living mammals using a bioluminescent", PHOTOCHEMISTRY AND PHOTOBIOLOGY, (1997) VOL. 66, NO. 4, PP. 523-531., XP000920570 *
CONTAG PR, OLOMU IN, STEVENSON DK, CONTAG CH.: "Bioluminescent indicators in living mammals.", NAT MED., vol. 4, no. 2, February 1998 (1998-02-01), pages 245 - 247, XP000914588 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8545814B2 (en) 1994-07-01 2013-10-01 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US6649143B1 (en) 1994-07-01 2003-11-18 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive localization of a light-emitting conjugate in a mammal
US7449615B2 (en) 1998-12-17 2008-11-11 Xenogen Corporation Non-invasive evaluation of physiological response in a transgenic mouse
US6610503B1 (en) 1999-03-17 2003-08-26 Xenogen Corporation Animal models for predicting sepsis mortality
US6964856B2 (en) 1999-03-17 2005-11-15 Xenogen Corporation Animal models and methods for sepsis
US6737245B1 (en) 1999-09-08 2004-05-18 Xenogen Corporation Luciferase expression cassettes and methods of use
US7196190B2 (en) 1999-12-16 2007-03-27 Xenogen Corporation Methods and compositions for screening for angiogenesis modulating compounds
US6867348B1 (en) 1999-12-16 2005-03-15 Xenogen Corporation Methods and compositions for screening for angiogenesis modulating compounds
US6647179B2 (en) 1999-12-21 2003-11-11 Agilent Technologies, Inc. Process and device for making gratings in optical fibres
US7056728B2 (en) 2000-07-06 2006-06-06 Xenogen Corporation Compositions and methods for use thereof in modifying the genomes of microorganisms
WO2002093143A2 (fr) 2001-05-17 2002-11-21 Xenogen Corporation Procede et appareil pour determiner la profondeur, la brillance et la dimension d'une cible dans une zone du corps humain
US7403812B2 (en) 2001-05-17 2008-07-22 Xenogen Corporation Method and apparatus for determining target depth, brightness and size within a body region
US8279334B2 (en) 2001-07-13 2012-10-02 Xenogen Corporation Structured light imaging apparatus
US7298415B2 (en) 2001-07-13 2007-11-20 Xenogen Corporation Structured light imaging apparatus
EP1373473A4 (fr) * 2001-12-04 2005-02-16 Genome Biosciences Llc Procedes et vecteurs de ciblage genetique
US7603167B2 (en) 2002-07-16 2009-10-13 Xenogen Corporation Method and apparatus for 3-D imaging of internal light sources
US7555332B2 (en) 2002-07-16 2009-06-30 Xenogen Corporation Fluorescent light tomography
US7599731B2 (en) 2002-07-16 2009-10-06 Xenogen Corporation Fluorescent light tomography
US7616985B2 (en) 2002-07-16 2009-11-10 Xenogen Corporation Method and apparatus for 3-D imaging of internal light sources
US8044996B2 (en) 2005-05-11 2011-10-25 Xenogen Corporation Surface construction using combined photographic and structured light information
WO2007026173A1 (fr) * 2005-09-02 2007-03-08 Birkeland Innovasjon As Modele d'animal transgenique pour identification de modulateurs de la proteine kinase
WO2007080621A1 (fr) * 2006-01-11 2007-07-19 Axxam S.P.A. Animaux transgéniques non humains luminescents, leur progéniture, leurs dérivés cellulaires et leurs applications
EP1808441A1 (fr) * 2006-01-11 2007-07-18 AXXAM S.p.A. Cellules souches luminescentes, mammifères transgéniques et leur utilisation
US10775308B2 (en) 2006-08-24 2020-09-15 Xenogen Corporation Apparatus and methods for determining optical tissue properties
US11730370B2 (en) 2006-08-24 2023-08-22 Xenogen Corporation Spectral unmixing for in-vivo imaging

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