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WO1992022670A1 - Detection precoce d'embryons transgeniques - Google Patents

Detection precoce d'embryons transgeniques Download PDF

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Publication number
WO1992022670A1
WO1992022670A1 PCT/US1991/004149 US9104149W WO9222670A1 WO 1992022670 A1 WO1992022670 A1 WO 1992022670A1 US 9104149 W US9104149 W US 9104149W WO 9222670 A1 WO9222670 A1 WO 9222670A1
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Prior art keywords
embryo
transgene
cell
restriction site
producing
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PCT/US1991/004149
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English (en)
Inventor
Paul Krimpenfort
Sang He Lee
Rein Strijker
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Genpharm International, Inc.
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Application filed by Genpharm International, Inc. filed Critical Genpharm International, Inc.
Priority to PCT/US1991/004149 priority Critical patent/WO1992022670A1/fr
Priority to AU22594/92A priority patent/AU2259492A/en
Priority to PCT/US1992/005097 priority patent/WO1992022647A1/fr
Publication of WO1992022670A1 publication Critical patent/WO1992022670A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • 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)

Definitions

  • transgenic cattle e.g., Wagner et al., Theriogenology 21:29-44 (1984); Logse et al., Theriogenology 23:205 (1985); all incorporated herein by reference.
  • a recent article has summarized the genetic engineering of livestock. (Pursel et al. Science 244:1281-1288 (1989) ; incorporated herein by reference) .
  • Methods for production of recombinant polypeptides in the milk of bovine species and methods for producing transgenic animals having desired phenotypes are discussed in co-pending U.S.S.N. 444,745 and 619,131; incorporated herein by reference.
  • transgenic livestock High costs and long gestation periods of large animals make it difficult, time consuming and expensive to produce transgenic livestock.
  • Current techniques to produce transgenic animals involve transfer of all microinjected embryos to recipients and identification of transgenic animals among newborns. Only 1.3% and 10% of the newborns were transgenic in sheep and pigs, respectively (Hammer et al., Nature 315:680-683 (1985), incorporated herein by reference), and 5% of bovine fetuses were transgenic (Biery et al., Theriogenology 29:224 (1988), incorporated herein by reference) . Since the frequency of transgene incorporation in mammals is often low, and their gestation time long, the detection of transgene integration, e.g.,in the preimplantation embryo would be highly desirable.
  • Methods for early detection of transgene integration would significantly decrease the number of pregnancies required to produce a transgenic animal and substantially increase the likelihood that an implanted embryo will produce a transgenic animal. These methods would thus allow for far more efficient production of transgenic offspring, providing significant savings of time and resources. Such methods would be especially important for those animals for which very low or non-existent frequencies of transgenesis have been obtained, e.g. bovine species.
  • the present invention fulfills these and other needs.
  • the present invention provides methods for the early detection of integrated transgenes in the nuclear genome of animal embryos.
  • integrated transgenes are detected by performing in situ hybridization on at least one metaphase stage cell from an embryo with a nucleic acid probe substantially complementary to the transgene.
  • the presence of such an integrated transgene is detectable as a hybridization signal on both sister chromatids of a chromosome in the metaphase spread.
  • Such a method is preferably nondestructive: a biopsy of at least one cell is removed from the embryo to be analyzed by in situ hybridization, and the resulting biopsied cell is viable.
  • the probe is typically labeled by biotinylation, and is preferably long, at least 2 kilobases in length to provide optimum sensitivity.
  • biotinylation In another method of early detection of transgene detection in animal embryos, one takes advantage of the differences in DNA methylation patterns in procaryotes and eucaryotes. Integration and replication of a transgene methylated in a procaryotic fashion causes it to become methylated in a eucaryotic fashion. Such a change can be tested for by use of methylation sensitive enzymes.
  • the restriction site of Dpn I is methylated in Dam * strains of E. coli or may be methylated jln vitro by dam methylase at N6 of an adenine of its restriction site. When integrated and replicated as part of the nuclear genome of an animal embryo, its restriction site loses its procaryotic methylation pattern, and can no longer be cleaved by Dpn I.
  • nuclear DNA from at least one cell from said embryo is treated with a restriction enzyme, such as Dpn I, capable of cleaving a methylated restriction site and incapable of cleaving said unmethylated restriction site, thereby producing restriction fragments of said nuclear DNA.
  • a restriction enzyme such as Dpn I
  • These restriction fragments are amplified by PCR with PCR primers substantially complementary to sequences on different strands of the transgene and flanking the unmethylated restriction site.
  • the amplified transgene may be detected only if the restriction enzyme has not cleaved its restriction sequence.
  • the embryos tested by these methods are typically bovine morulas at the 8 to 16 cell stage. It is preferable to clone the animal embryo prior to testing for an integrated transgene, such as by nuclear transfer. Methods are also provided which additionally determine the sex of the embryo tested by the methods above described. Also embraced are embryos which have been produced by any of the methods of the present invention.
  • the present invention provides methods for early detection of transgene integration in embryos which permit implantation and development to maturity of transgenic embryos so analyzed.
  • Two basic approaches are provided: the first is based on the polymerase chain reaction (PCR) , the second on the 'in situ 1 hybridization of nuclear DNA from embryonic cells with labeled probes. Each is described below in detail. These not only allow the early and rapid detection of transgene sequences in the cells of embryos into which transgene DNA has been introduced, but allow one to determine whether such DNA is truly integrated into the nuclear genome, giving rise to a transgenic animal, or is nonintegrated DNA which will eventually be lost.
  • PCR polymerase chain reaction
  • False positives arising when nonintegrated DNA is mistakenly identified as integrated transgenes, would otherwise demand a significant expenditure of time and resources.
  • the power of these techniques is enhanced when they are used in concert, preferably using PCR as an initial detection method, followed by in situ hybridization as a confirmatory method.
  • Transgene sequences may be introduced into host cells in a variety of ways well known in the art, including: electroporation (Thomas and Capecchi, Cell 51:503-512 (1987) or microinjection of the transgene into the pronuclei of fertilized oocytes or nuclei of ES cells of the animal (Zimmer and Gruss, Nature 338:150-153 (1989); transfection of ES cells in culture by calcium phosphate precipitation (Gossler et al., Proc. Natl. Acad. Sci. USA 83:9065-9069 (1986); and infection of zygotes with a retrovirus containing the transgene (Jaenisch, Proc. Natl. Acad. Sci. USA, 73. 1260-1264 (1976) ; Robertson et al., Nature 323:445-448 (1986); all incorporated herein by reference) .
  • pre-implantation embryos preferably contain approximately 16 to 150 cells.
  • the 8 to 32 cell stage of an embryo is commonly referred to as a morula.
  • blastocysts Those pre-implantation embryos containing more than.32 cells are commonly referred to as blastocysts.
  • Blastocysts are generally characterized as demonstrating the development of a blastocoel cavity, typically at the 64 cell stage.
  • Methods for culturing fertilized oocytes to the pre-implantation stage include, for example, those described by Gordon et al., Meth. Enzymol.
  • a biopsy is obtained, i.e., at least one cell is removed from the embryo.
  • the embryo is preferably not cultivated past the morula stage (32 cells) .
  • Division of the pre-implantation embryo may result in two "hemi-embryos" (hemi-morula or hemi-blastocyst) , at least one of which is viable, i.e., capable of subsequent development after implantation into the appropriate female to develop in utero to term.
  • an embryo may be either equally or unequally divided into two hemi-embryos which are not necessarily of equal cell number. Indeed, only one or two cells may constitute a biopsy from a single embryo; for that reason, one may more accurately speak of a "biopsied embryo” resulting from the removal of a biopsy of one or more cells from an embryo for analysis by the early detection methods provided herein. Essentially, all that is required is that one of the embryos which is not analyzed as hereinafter described be of sufficient cell number to develop to full term in utero.
  • the biopsied embryo if shown to be transgenic, may be implanted in a female recipient animal to develop to term.
  • the methods for detecting transgenesis in pre-implantation embryos provided herein are combined with embryonic cloning steps to generate a population of transgenic embryos having the same genotype, several of which may provide cell biopsies for transgenesis detection.
  • embryonic cloning steps to generate a population of transgenic embryos having the same genotype, several of which may provide cell biopsies for transgenesis detection.
  • such cloned embryos may thereafter be implanted into recipient females to produce a population of transgenic animals also having the same genotype.
  • embryonic cloning ensures not only that enough biopsied cells are available to provide a conclusive test for transgenesis, but also that at least one of the cloned embryos shown to be transgenic will produce a transgenic animal.
  • embryo cloning may be performed by several different approaches. In one cloning method, the transgenic hemi-embryo is cultured in the same or in a similar media as used to culture individual oocytes to the pre-implantation stage.
  • transgenic embryo so formed (preferably a transgenic morula) is then divided into "transgenic hemi- embryos" which can then be implanted into a recipient female to form a clonal population of two transgenic animals.
  • transgenic hemi-embryos obtained may be again cultivated to the pre-implantation stage, divided, and recultivated to the transgenic embryo stage. This procedure is repeated until the desired number of clonal transgenic embryos having the same genotype are obtained.
  • Such transgenic embryos may then be implanted into recipient females to produce a clonal population of transgenic animals.
  • the transgenic embryo is cloned by nuclear transfer according to the techniques of Prather et al. (Biol. Reprod. 37:59-86 (1988), incorporated herein by reference) and Roble et al. (J. Anim. Sci. 64:642-664 (1987) ; incorporated herein by reference) .
  • nuclei from individual cells of the transgenic embryo are transplanted into enucleated oocytes, each of which is thereafter cultured to the blastocyst stage.
  • the transgenic embryos may be subjected to another round of cloning or may be transferred to a recipient parent for production of transgenic offspring having the same genotype.
  • one may produce multiple, genetically identical embryos.
  • One may obtain one or a small number of cells from each of these identical embryos to test for transgenesis by the methods of the present invention.
  • transgenesis is understood herein as meaning the integration of a transgene into the nuclear genome of a host organism, and its subsequent replication and stable maintenance as part of the nuclear genome.
  • integration is meant that the transgene is located on a chromosome, continuous with and linked by covalent bonds to the double stranded DNA which constitutes the chromosome. This situation may be contrasted with that of non-integrated extra-chromosomal DNA elements such as plasmids, episomes, and double minutes.
  • transgenic embryos and/or transgenic animals having the same "genotype” means that the genomic DNA is substantially identical among the individuals of the embryo and/or transgenic animal population, having derived from the same genetic source. During mitosis, however, various somatic mutations may occur which may produce some variations in the genotype of one or more cells and/or animals. Thus, a population considered to have the same genotype may demonstrate individual or subpopulation variation. Transgene detection using PCR
  • PCR methods are well known and widely practiced in the art. See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202, and PCR Protocols: A Guide to Methods and Applications f Innis et al. eds., Academic Press, San Diego (1990) , all incorporated herein by reference. Reagents and hardware for conducting PCR are commercially available through Perkin-Elmer/Cetus Instruments (PECI) of Norwalk, Connecticut.
  • PECI Perkin-Elmer/Cetus Instruments
  • PCR is a cyclical process for the amplification of a nucleic acid template comprising the steps of: (a) denaturation of the template (generally by heating to 95 ⁇ C to 100°C), (b) hybridization of dual oligodeoxynucleotide primers to the denatured template, and (c) template replication, consisting of an extension of these primers by a DNA polymerase (generally a thermostable polymerase such as Taq polymerase) .
  • a DNA polymerase generally a thermostable polymerase such as Taq polymerase
  • dNTPs deoxynucleotide triphosphates
  • a buffer to provide, for example, the salt and pH conditions necessary for optimal polymerase activity.
  • the primers are so designed that they hybridize to sequences flanking the region to be amplified, one on each strand, and that the single strand generated by extension of each primer can act as a template for the extension of the other primer.
  • each cycle of replication provides a two-fold amplification of the template.
  • PCR can be thus be used to amplify target DNA sequences several million-fold.
  • the amplified DNA can then be analyzed by restriction enzymes and electrophoresis.
  • oligonucleotides primers are synthesized that preferably hybridize specifically to sequences in the transgene, or, if present, to sequences in accompanying vector sequences. Upon amplification of the target sequence a DNA fragment of specific size will be generated.
  • a typical PCR buffer contains 10 to 50 mM Tris-HCl
  • this buffer may be necessary for certain combinations of target nucleic acids and primers, as will be readily appreciated by those skilled in the art.
  • DNA polymerases are appropriate for performing PCR.
  • Thermostable polymerases are preferred, such as those obtained from Thermus a uaticus (Taq polymerase) or T. flavis.
  • Taq polymerase Thermus a uaticus
  • T. flavis T. flavis
  • other polymerases may be appropriate, including E. coli DNA polymerase I or its Klenow fragment, reverse transcriptase, phage T4 or T7 polymerases, or structural variants and modified forms of these and other polymerases.
  • PCR The sensitivity of PCR is particularly useful for analyzing small DNA samples (e.g. from a small number of cells) .
  • This sensitivity can, however, be problematic unless stringent precautions are taken to avoid specific contamination.
  • DNA contamination from aerosol particles, pipettes, primers, etc. can be a big problem when the DNA used for injection of the embryos is prepared in the same lab where PCR is performed.
  • PCR cannot discriminate between integrated DNA and non-integrated DNA.
  • stringent measures can prevent contamination: PCR analyses in a completely isolated lab equipped with a flow hood with filter; special equipment (pipettes, microfuges, etc.) dedicated to PCR; all reagents (primers, buffers, Taq polymerase) supplied in kits that can be opened and used only once.
  • the problem of discriminating integrated from non ⁇ integrated DNA constructs may be solved by taking advantage of the difference in methylation patterns between DNA replicated in procaryotes and DNA replicated in eucaryotes.
  • DNA constructs which have been cloned in procaryotes and/or methylated in vitro will retain the procaryotic methylation profile, unless they integrate in and, consequently, are replicated as part of the host genome.
  • the A of the sequence GATC the recognition site for the restriction enzyme Dpn I
  • Dpn I is methylated in Dam * strains of E. coli; it is unmethylated in eukaryotic cells, which lack the dam methylase.
  • Eukaryotic DNA is methylated at G in the sequence GC.
  • the integrated transgene After several cleavages, the integrated transgene, having been replicated along with the nuclear genome, will be methylated in a eucaryotic fashion.
  • restriction endonucleases capable of cutting methylated restriction sites but not nonmethylated restriction sites (e.g., Dpn I)
  • restriction endonuclease resistant, DNA available for amplification.
  • only embryos having an integrated transgene should give rise to an amplified band.
  • the use of such a restriction enzyme also reduces the problem of contamination by bacterial DNA (e.g., plasmid) contamination.
  • methylation sensitive enzymes which can cut sequences in the transgene which display the eukaryotic methylation pattern but which fail to cut the same sequences when they display a prokaryotic methylation pattern.
  • inverse PCR An alternative method for detecting integrated transgenes, "inverse PCR", makes use of primers which have opposite (reverse) orientation (Triglia et al., Nucl. Acids Res. 16:8186 (1988); Ochman et al., Genetics 120:621-623 (1988) , both incorporated herein by reference) .
  • the DNA isolated from biopsies is cut with restriction enzymes and the fragments are circularized by ligation.
  • DNA fragments of expected length are amplified (derived from concatamers)
  • DNA from transgenic embryos will also give rise to fragments of varying size. These fragments contain host sequences which are present between the reverse primers in the religated circles.
  • transgene may be quite long (up to 50 kb or more, and typically 20 kb or more) and often lack vector sequences
  • PCR primers are preferably designed to be complementary to sequences within the transgene itself.
  • a preferred method for detecting transgenesis by PCR at an early stage in the embryo's development employs a methylation sensitive restriction endonuclease such as Dpn I.
  • Dpn I recognizes and cleaves the sequence GATC in double stranded DNA only when the adenine in each strand within this sequence is methylated at N-6.
  • the transgene containing the sequence GATC is methylated prior to microinjection either by transferring the transgene on an appropriate plasmid to a Dam * strain of a microorganism such as E. coli MM294, or by directly methylating the transgene in vitro with dam methylase (commercially available from a number of vendors, including New England Biolabs) .
  • the methylated transgene (preferably without any exogenous sequences such as plasmid vector) is then microinjected into fertilized oocytes (approximately 10 to 500 copies per pronucleus, more preferably 50 to 100 copies per pronucleus) .
  • fertilized oocytes so obtained are cultured in vitro to the pre-implantation stage. During this early growth and cell division phase, the genomic DNA is replicated. Accordingly, those copies of the methylated transgene integrated into the genome of the fertilized oocyte are unmethylated after replication, whereas any non-integrated transgenes which may still exist after replication will remain methylated (Lacks et al., J. Mol. Biol. 114:153 (1977), incorporated herein by reference) .
  • This differential methylation pattern for an integrated versus a non-integrated transgene permits the identification of which fertilized oocytes have integrated the transgene into the genome.
  • the identification of the pre-implantation embryos containing the integrated transgene is achieved by analyzing the DNA from each of the embryo biopsies (or the pooled biopsies from several genetically identical embryos arising from cloning a single embryo) .
  • DNA is typically obtained by lysing the biopsied cells and analyzing the released DNA af er treatment as described by Ninomiya et al., Molecular Reproduction and Development 1:242-248 (1989), incorporated herein by reference.
  • Each of the DNA samples is treated with Dpn I. Thereafter, PCR is used to amplify all or part of the transgene.
  • extension primers each complementary to opposite strands at opposing ends of the transgene are used.
  • such extension primers are chosen such that the amplified gene product spans the Dpn I site in the transgene. That is, the primer binding sites for the two PCR primers flank the Dpn I site. If Dpn I cleavage has not occurred, PCR amplification will result in amplified sequences having a predicted size, whereas no amplification product will result for those transgenes which have been cleaved.
  • the Dpn I digested and PCR amplified DNA from the biopsy is subjected to electrophoresis.
  • electrophoresis is preferably followed by hybridization with labeled probe complementary to the region of the transgene between the two extension primers.
  • size of the amplified DNA sequences if any, is determined.
  • the presence of an amplified sequence of the appropriate size indicates whether the transgene has been integrated into the pre-implantation embryo from which the biopsy was obtained (now called a "transgenic biopsied embryo") .
  • the remaining untreated transgenic biopsied embryo is transplanted into a recipient parent.
  • the transgenic animal having the desired phenotype conferred by the integrated transgene is identified by an appropriate method in utero or after birth.
  • Dpn I requires that the sequence GATC be present in the transgene of interest. In those cases when such a sequence is not present, it may be readily introduced into the transgene by site directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985), incorporated herein by reference) or cassette mutagenesis (Wells et al., Gene 34:315 (1985), incorporated herein by reference) , provided such mutagenesis does not change the amino acid sequence encoded by the transgene (or causes an inconsequential change in amino acid sequence) and that any codons so generated are functional in the transgenic animal of interest.
  • transgenic embryos and/or transgenic animals having the same "genotype" means that the genomic DNA is substantially identical between the individuals of the embryo and/or transgenic animal population. It is to be understood, however, that during mitosis various somatic mutations may occur which may produce some variations in the genotype of one or more cells and/or animals. Thus, a population having the same genotype may demonstrate individual or subpopulation variations.
  • a sample is electrophoresed on an agarose or polyacrylamide gel.
  • Smaller DNA fragments resulting from amplification are preferably analyzed by polyacrylamide gel electrophoresis.
  • DNA bands are visualized by ethidium bromide staining, or, to increase sensitivity, gel electrophoresis can be followed by Southern blotting or dot blotting and hybridization with a labeled probe by techniques well known in the art.
  • labeled PCR primers which are incorporated into the amplified DNA product, may be employed by methods well known in the art to assist in visualization of the amplification product and thus increase sensitivity.
  • nucleic acid probes are used to locate specific complementary nucleic acid sequences, e.g., specific DNA sequences on intact chromosomes, in situ, a procedure called "in situ hybridization".
  • Labeled nucleic acid probes of a predetermined nucleotide sequence are hybridized to the chromosomes of sample cells (or tissues) , the DNA double strands of which have been denatured by a brief exposure to a very high pH.
  • the chromosomal regions that bind the probe during the hybridization step are examined by microscopy to determine whether they hybridize to the probes and thus contain the specific nucleic acids of interest.
  • the direct methods the reporter molecule is bound to the nucleic acid probe so that the molecular hybrids between probe and target sequences can be visualized microscopically immediately after the in situ hybridization procedure.
  • Such methods include the terminal fluorochrome labeling procedure of RNA probes (Baumann et al., 1980, 1984) and the direct enzyme labeling procedure of nucleic acids (Renz and Kurz (1984) .
  • the probe must contain an element, introduced chemically or enzy atically, that renders it detectable by affinity cytochemistry, hence the term indirect.
  • oligonucleotides containing functional groups i.e. primary aliphatic amines or sulfhydryl groups
  • haptens e.g., biotin or digoxygenin
  • reporters like fluorochromes or enzymes
  • the probes are synthesized with nucleotides that contain a biotin side chain, and the hybridized probes are detected by staining with a network of streptavidin and some type of marker molecule.
  • this technique it is possible to detect specific, single copy nucleic acid sequences in individual cells and on individual chromosomes (See for review: Raap et al., In Techniques in Immunocvtochemistrv, vol. IV, ed. G. Bullock and P. Petrusz: New York, Academic Press (1990) , incorporated herein by reference) .
  • RNA probes labeled directly with a terminal fluorochrome labeled directly with a terminal fluorochrome.
  • the sensitivity of these direct methods was low. Therefore, immunocytochemical amplification procedures have been developed to increase detection sensitivity.
  • Several chemical and enzymatic nucleic acid hapten modifications are now available for the amplification on DNA or RNA probes for in situ hybridization (Raap et al., 1989). All modifications have in common that the hapten does not affect the hybridization properties of the probes. Most haptenized probes are detected, after hybridization, with antibodies specific for the hapten.
  • in situ hybridization is a fairly complex multistep procedure in which each step must be optimized, the technique has been successfully employed, for example, in the diagnosis of viral infection (Brigati et al., Virology 126:32- 50 (1982); Burns et al. , J. Clin. Pathol. 39:1066-1073 (1986); Raap et al., Histochemistry 88:367-373 (1988); all incorporated herein by reference) and the analysis of gene expression at the mRNA level (Rudkin and Stollar, Nature 265:472-473 (1977), incorporated herein by reference) ; for the assessment of chromosome copy number in early prenatal diagnosis (Julien et al.
  • PCR will be employed to provide an initial determination whether the transgene is incorporated into the genome of an embryo, while in situ hybridization is used to confirm the results of the initial testing.
  • PCR may be used to confirm the results obtained by an initial test using in situ hybridization.
  • the sex of the embryos so tested may further be sexed by PCR or in situ hybridization using probes specific for the Y chromosome, or by other methods well known in the art.
  • in utero analysis is performed by several techniques.
  • transvaginal puncture of the amniotic cavity is performed under echoscopic guidance (Bowgso et al. (1975) Bet. Res. 96:124-127; Rumsey et al. (1974) J. Anim. Sci. 39:386-391; both incorporated herein by reference) .
  • This involves recovering about 15 to 20 milliliters of amniotic fluid between about day 35 and day 100 of gestation.
  • This volume of amniotic fluid contains about 1000 to 12,000 cells per ml originating from the urogenital tract, the skin, and possibly the lungs of the developing embryo. Most of these cells are dead. Such cells, however, contain genomic DNA which is subjected to PCR analysis for the transgene as an indication of a successful transgenesis. Alternatively, fetal cells may be recovered by chorion puncture. This method also may be performed transvaginally and under echoscopic guidance. In this method, a needle is used to puncture the recipient animal's placenta, particularly the placentonal structures, which are fixed against the vaginal wall. Such sampling may be performed around day 60 of gestation. Chorion cells, if necessary, are separated from maternal tissue and subjected to PCR analysis for the transgene as an indication of successful transgenesis.
  • Transgenesis may also be detected after birth.
  • transgene integration can be detected by taking an appropriate tissue biopsy such as from the ear or tail of the putative transgenic animal. About one to two centimeters of tail or about five to ten square millimeters of ear are obtained, followed by Southern blotting with a probe for the transgene according to the method of Hogan et al. (1986)
  • Recombinant DNA techniques It will be readily apparent that the present invention relies heavily upon a thorough knowledge and understanding of recombinant DNA technology.
  • the recombinant DNA techniques employed when using the present invention are well established and constitute recognized methods, e.g., for the use of restriction endonucleases; for the preparation of predetermined nucleotides in sequence as hybridization probes; and for the various methods of labelling such DNA or RNA probes using a variety of labels, such as radionuclides. Accordingly, it is presumed that one practicing the present invention is familiar with the applications and limits of the various techniques and will recognize that minor changes in reagents, concentrations, temperature, reaction times and similar alterations of known methods are merely obvious variations of choice.
  • transgene refers to a DNA sequence which is capable of producing a desirable phenotype when contained in the genome of cells of a transgenic animal. Such a transgene often comprises a recombinant DNA sequence encoding a "recombinant polypeptide". In such cases, the transgene is capable of being expressed to produce the recombinant polypeptide. Unless otherwise noted, the term also is used to embrace vector sequences and non-coding flanking sequences which may accompany such polypeptide-encoding sequences.
  • the PCR primers used in the methods of the present invention are oligonucleotides, whether occurring naturally as in a purified restriction digest, or produced synthetically, which are capable of hybridizing specifically to a known sequence in a target gene.
  • a PCR primer especially the terminal 3' nucleotide of the primer, has hybridized, it acts as a point of initiation of synthesis under conditions in which synthesis of a primer extension product is favored.
  • Such conditions typically include the presence of four different nucleotide triphosphates and a thermostable polymerase in an appropriate buffer and at a suitable temperature.
  • Such primers are preferably single stranded for maximum efficiency and amplification, although double stranded primers may be employed if treated to separate the complementary strands before use.
  • the oligonucleotides employed as primers may contain naturally occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine, and may be either DNA or RNA.
  • Such an oligonucleotide will preferably contain at least one or preferably more than one region of eight or more consecutive nucleotides having perfect complementarity with the target sequence. They will also usually have one or more nucleotides at the 3' end displaying perfect complementarity (i.e., base pairing) with a single stranded region of a target nucleic acid. However, longer oligonucleotides (e.g., more than 20 nucleotides) may anneal with the desired specificity, even though no such region of consecutive nucleotides with perfect complementarity may be present.
  • the primer sequence need not, however, reflect the exact sequence of the template, and may include sequences in addition to those allowing the primer to hybridize with specificity to the template. Alternatively, noncomplementary bases or longer sequences can be interspersed into the primer provided that the primer sequence has sufficient complementarity with the sequence of the target sequence to hybridize with it and thereby allow synthesis of the extension product.
  • the primer may include nucleotides which have been substituted, e.g., with biotin. As a consequence of amplification by PCR, the sequence and substituents of the primer are introduced into the amplified product.
  • primer sequences are preferably synthesized using commercially available methods and equipment.
  • Synthetic oligonucleotides can be produced by the solid phase phosphora idite method according to Caruthers et al., Cold Spring Harbor Svmp. Quant. Biol. ,
  • oligonucleotide is an oligodeoxyribonucleotide. Its exact length will depend on such factors as number of mismatches, if any, temperature, salt conditions, and other parameters discussed above. Oligonucleotide primers typically are from about 8 to 50, usually about 12 to 50, and preferably 16 to 30 nucleotides in length, although longer or shorter primers may be appropriate.
  • Suitable probes may be RNA or DNA and may be either double stranded or single stranded.
  • In situ hybridization probes will preferably be as long as possible (greater than 2 kb is preferable) to increase sensitivity, although shorter probes may be used, and will preferably be complementary to exon sequences in a transgene to reduce the chance for nonselective hybridization.
  • Probes for Southern blots may be shorter probes substantially complementary to sequences in a transgene.
  • the nucleic acid probes may be derived from genomic DNA or cDNA, prepared by chemical or enzymatic synthesis (e.g., as an RNA transcribed from a template sequence comprising a sequence substantially complementary to a transgene) , or may be a hybrid of the various combinations. Recombinant nucleic acids comprising sequences otherwise not naturally occurring may also be employed. They may contain naturally occurring nucleotides or their analogs, such as 7-deazaguanosine or inosine.
  • probes may be labeled by any of the methods commonly used in the art, such as nick translation or random hexamer labeling.
  • nucleic acid probes will include an isolated nucleic acid attached to a label or reporter molecule.
  • Probes may be prepared by nick translation, Klenow fill-in reaction, random hexamer priming, or other methods known in the art. For isolating nucleic acids, choosing label or reporter molecules, labeling probes, and other aspects of probe preparation see, inter alia. Sambrook et al.. Molecular
  • An oligonucleotide primer or a probe is functionally defined as "substantially homologous" or “substantially complementary” to a target nucleic acid when it will anneal or hybridize to a single desired position on a strand of the nucleic acid being targeted or its complementary strand such that stable and specific binding occurs between the primer and the target nucleic acid under selective conditions (See, M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference) . Selectivity of hybridization exists when hybridization occurs which is more selective than total lack of specificity, and is generally marked, in the methods of the present invention, by the formation of a single desired extension product.
  • Proper annealing conditions depend, for example, upon an oligonucleotide*s length, base composition, and the number of mismatches and their position on an oligonucleotide, or on the annealing temperature, salt concentration of the medium, and other conditions, and must often be determined empirically.
  • oligonucleotide design and annealing conditions see, for example in Sambrook et al. (1989) or F. Ausubel et al., ed. (1987), which are incorporated herein by ref rence.
  • Stringent primer annealing conditions will vary with the specific application, but typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM.
  • Temperature conditions will typically be greater than 22 ⁇ C, more typically greater than about 30°C and preferably in excess of about 37 ⁇ C. As other factors may dramatically affect the stringency of hybridization, including base composition and size of the complementary strands, the presence of such salts as MgCl , and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
  • an oligonucleotide primer or a probe is considered “substantially homologous” or substantially complementary" to a target sequence whenever the primer and target sequence, or their complementary strands, when optimally aligned and compared, are identical with appropriate nucleotide insertions or deletions, generally in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98 to 99.5% of the nucleotides.
  • Immature oocytes are obtained in large quantity (400- 600/day) by aspirating follicles of ovaries obtained at abbatoirs. Immature oocytes are cultured for a period in vitro before they are competent to be fertilized. Once "matured", oocytes are fertilized with sperm which has also been matured, or "capacitated” in vitro. The pronuclei of the fertilized oocyte is then injected with the transgene encoding for the expression and secretion of human lactoferrin.
  • Zygotes resulting from this in vitro fertilization and microinjection are then cultured to the late morula or blastocyst stage in media with somatic tisssue, or in medium "conditioned” by somatic tissue. Blastocysts are then transferred non- surgically to recipient cattle for the balance of gestation or analyzed for integration of the transgene as described herein.
  • IMM In vitro maturation
  • Ovaries are obtained immediately after slaughter at local abbatoirs and oocytes are recovered.
  • oocytes are obtained from living cattle by surgical, endoscopic, or transvaginal ultrasonic approaches. In all cases, oocytes are aspirated from ovarian follicles (2-10 mm diameter) . After washing, oocytes are placed in a maturation medium capable of supporting nuclear and cytoplasmic maturation of bovine oocytes. Examples of such media are given by Sirard et al. (Biol. Reprod. 39:546-552 (1988) , incorporated herein by reference) .
  • IVF In vitro fertilization
  • Matured oocytes are fertilized with either fresh or frozen thawed sperm.
  • Sperm are then added to a fertilization media consisting of a modified Tyrode's solution (Parrish et al. (1986) supra.. incorporated herein by reference) supplemented with heparin to induce sperm capacitation (Parrish et al., Biol. Reprod. 38:1171-1180 (1988) , incorporated herein by reference) .
  • Capacitation constitutes the final sperm maturation process which is essential for fertilization.
  • Sperm and oocytes are co-cultured for 18 hours.
  • a useful feature of this IVF method is that (in the case of frozen sperm) consistent, repeatable results are obtained once optimal fertilization conditions for a particular ejaculate have been defined (Parrish et al. (1986) supra. , incorporated herein by reference) .
  • IVC In vitro culture
  • Conventional culture systems which support development of mouse, rabbit, or human ova, do not support development of bovine embryos past the 8-16 cell stage.
  • This problem has been overcome by pre-conditioning culture media with oviductal tissue. Oviduct-conditioned medium will support bovine embryos past the 8-16 cell stage to the blastocyst stage in vitro (Eyestone and First, J. Reprod. Fert. 85:715-720 (1989), incorporated herein by reference).
  • Bovine embryos have proven refractory to in vitro culture. This in part stems from the existence of a "block" to cleavage in vitro at the 8-16 cell stage.
  • This block may be alleviated by culturing embryos in the oviducts of rabbits (reviewed by Boland, Theriogenology 21:126-137 (1982), incorporated herein by reference) or sheep (Willadeen in: Mammalian Egg Transfer (E. Adams, ed.), pp. 185-210 (1982); Eyestone et al. , Theriogenology 28:1-7 (1987); both incorporated herein by reference) .
  • Bovine embryos did not yield to attempts to culture them in vitro past the 8-16 cell "block” until Camous et al., J. Reprod. Fert. 72:479-485 (1984), incorporated herein by reference) demonstrated cleavage to 216 cells when embryos were co-cultured with trophoblastic tissue.
  • Blastocysts have been produced in this system after superovulation and artificial insemination, or by in vitro maturation (IVM) , and fertilization (IVF) of immature oocytes. Blastocysts produced in this fashion resulted in pregnancies and live calves after transfer to recipient animals. The results obtained were as follows:
  • Oviduct Tissue Preparation of Oviduct Tissue and Use For Co-Culture and Conditioned Medium.
  • Ovine oviducts are obtained after slaughter or by salpingectomy.
  • the lumenal tissue is harvested by scraping an intact oviduct gently with a glass slide and washed five times in 10 ml modified tyrodes-hepes solution (Parrish et al., Biol. Reprod. 38:1171-1180 (1988), incorporated herein by reference) .
  • the final tissue pellet is suspended in M199 + 10% fetal calf serum at a ratio of l volume tissue to 50 volumes of media.
  • the tissue suspension can be used for embryo-co- culture.
  • media may be conditioned for 48 h, and after centrifuging the suspension, the supernatant may be used as embryo culture medium.
  • Conditioned medium may be stored at -70 ⁇ C, if desired.
  • Conditioned medium should be used at full strength for embryo culture (no dilution) (Eyestone (1989)).
  • the DNA fragment containing the hLF expression unit is excised from the vector by digestion with the appropriate restriction enzyme(s) and separated by agarose gel electrophoresis. The fragment is purified by electroelution, phenol and chloroform extraction and ethanol precipitation. The DNA fragment is dissolved in and dialyzed in 10 mM Tris, 0.1 mM EDTA (pH 7.2) at a concentration of 1 to 2 ⁇ g/ml. Microinjection needles are filled with the dialyzed DNA solution.
  • Bovine pronuclei are injected in the same manner as murine pronuclei (Hogan et al., in: Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory (1986) , incorporated herein by reference) with an additional centrifugation step in order to visualize the pronuclei.
  • the injection takes place 18-24 hours after fertilization. The time varies depending on the bull used as a source of semen. Nuclei become visible at different times in different batches of semen.
  • Bovine oocytes matured and fertilized in vitro, are spun in a microfuge tube (Eppendorf) in 1 ml of tyrodes-hepes solution (Parrish (1987) , incorporated herein by reference) at 14500xg for eight minutes (Wall et al., Biol. Reprod. 32:645- 651 (1985) , incorporated herein by reference) .
  • the embryos are transferred to a drop of tyrodes-hepes solution on a microscope slide covered with paraffin oil.
  • Using a hydraulic system the oocytes are fixed to the egg holder in such a way that both the pronuclei are visible (using interference-contrast or phase contrast optics) .
  • the oocytes are rolled to change their position on the egg holder to visualize the pronuclei.
  • the injection needle is brought into the same sharp focus as one of the pronuclei.
  • the needle is then advanced through the zona pellucida and cytoplasm into the pronucleus.
  • a small volume, 1-3 pi, is injected (containing 20-100 DNA copies) into the pronucleus either by using a constant flow or a pulse flow (using a switch) of DNA solution out of the needle.
  • two cell stage embryos are spun as described and the nuclei of both blastomers are injected as described.
  • the injected embryos are then transferred to a drop of co-culture medium as described in Example 1 in order to develop to the morula or blastocyst stage.
  • oviduct cell conditioned medium was compared with oviduct epithelial cell coculture system as systems for culturing in vitro matured and fertilized oocytes up to the blastocyst stage.
  • oviduct epithelial cell coculture system embryos are cultured on a monolayer of bovine oviduct epithelial cells (BOEC) . Using this system, 30% of the fertilized oocytes develop to the blastocyst stage.
  • BOEC bovine oviduct epithelial cells
  • the isolation of single or multiple blastomers from a preimplantation embryo requires penetration of the zona pellucida (ZP) .
  • ZP zona pellucida
  • the urine ZP can easily be dissolved at a precise, limited area using a constant narrow flow of acidic Tyrode (pH 2.3) solution, although the ZP of bovine embryos cannot be dissolved by acidic Tyrode, even with a pH lower than 2.1.
  • a slit in the ZP and aspirating blastomers is made in the ZP which can be opened with a beveled glass pipet with an opening large enough to subsequently aspirate blastomers. After withdrawing the aspiration pipet the ZP will reclose. This method is more laborious but can be more precisely controlled and causes the least disturbance of normal development. Little damage is caused to the embryo and to the blastomers that are removed.
  • Biopsies obtained as described herein are also useful for multiplication of embryos proven to be transgenic.
  • the oocyte Upon the microinjection of a construct, the oocyte is cultured, preferably at least to the 8-cell, even though analysis of embryos before that time is possible.
  • a proper site of each embryo is cleaved and subjected to lysis (King et al.. Molecular Reprod. and Devel. 1:57-62 (1988); proteolysis (Higuchi, "Amplifications (A Forum for PCR Users” 2:1-3 (1989); both incorporated herein by reference) and Dpn I digestion.
  • PCR is performed as described previously (Ninomiya et al., Molecular Reprod. and Devel. 1:242-248 (1989), incorporated herein by reference) with sets of two primers which flank a Dpn I site in the construct.
  • One such set of primers are the forward primer ATG AAA CTT ATC CTC ACC TGT CTT GTG (in the ⁇ Sl portion) and the reverse primer GGG TTT TCG AGG GTG CCC CCG AGG ATG GAT (in the hLF portion) of an hLF transgene disclosed in the commonly owned co-pending application U.S.S.N. 619,131, incorporated herein by reference.
  • the following buffer is added to a total volume of 50 ⁇ l in a 500 ⁇ l microfuge (Eppendorf) tube: 50 mM KC1, 10 mM Tris-HCl (pH 9.0 at 25°C) , 1.5 mM MgCl 2 , 0.01% gelatin, 0.1% Triton X-100, 50 ⁇ M each of dATP, dGTP, dCTP and dTTP, 50 pmol forward primer, 50 pmol reverse primer, 0.8 U Dpnl (Bethesda Research Laboratories) , and 2 U Taq polymerase (Promega) .
  • the mixture is overlaid with a drop of light mineral oil to prevent evaporation.
  • the mixture is incubated at 37°C for 20 min, followed by 93°C for 5 min. 50 cycles of PCR follow with each cycles consisting of: denaturation for 1 min at 93°C; annealing for 1 min at 55 ⁇ C; and extension for 1.3 min at 72°C. After the 50 cycles are complete, a further 10 min incubation at 72 ⁇ C follows. The reaction may be held at 4°C at this stage until further analysis is undertaken.
  • To analyze the PCR amplified products about one- third to one-half of the PCR reaction volume is electrophoresed on an agarose or polyacrylamide gel. DNA bands are visualized by ethidium bromide staining, or, if additional sensitivity is required, by Southern blotting of the PCR products followed by probing with a labeled probe substantially complementary to the transgene.
  • Fixation Three/one, methanol/glacial acetic acid) is freshly prepared and kept at -20°C until needed. Three ml of precooled fixative is placed in a precooled thick watch glass and the embryos are added. It is important that the watch glass is thick enough to maintain the temperature of the fixative reasonably constant during ensuing operation under the microscope. If the temperature rises quickly, two problems arise: (1) most of the embryos will burst, (2) the embryos do not remain sunken at the bottom of the watch glass but float around in the fixative, making it impossible to find and collect them under the microscope. The fixation time is approximately 30 minutes at -20°C.
  • RNAse treatment Before the initiation of hybridization procedure an individual metaphase spread on each slide is located by phase- contrast microscopy and recorded for speedy detection of the metaphase spread after the in situ hybridization procedure.
  • RNAse treatment Each slide is incubated with 100 ⁇ l RNAse (0.1 mg/ l) under a coverslip for 60 minutes at 37 ⁇ C in a slide-jar. At the end of incubation, the coverslip is removed by the addition of 100 ml of 2xSSC (Sambrook et al., 1989) and gentle shaking of the slide-jar for 5 minutes on the top of an automatic shaker. The slides are further washed three times with 2xSSC at room temperature. Throughout the entire procedure, all the washings require 5 minutes incubation at room temperature with constant gentle shaking unless specified otherwise. Slides are dehydrated by sequential washing with 70%, 90%, and 100% ethanol.
  • denaturation solution a mixture of 70 ⁇ l of 100 formamide, 10 ⁇ l of 20xSSC and 10 ⁇ l of 0.5 M phosphate, pH 7.0 and 10 ⁇ l of H O
  • Probe Preparation One ⁇ g of DNA for the probe is digested with DNase (0.001 ng/ ⁇ l) overnight at 370 ⁇ C, then nick-translated in the presence of 40 ⁇ M biotin-11-dUTP (Sigma) for 1 hour at 14 ⁇ C. The reaction is stopped by addition of 0.5 M EDTA. The labelled prove is purified by running on a G-50 sephadex column. The degree of labelling of the probe is monitored by using BluGENE (BRL) , a nonradioactive nucleic acid detection system.
  • DNase 0.001 ng/ ⁇ l
  • Probe (20 ng per slide) and sheared bovine genomic DNA (10 ⁇ g per slide) is dissolved in a desired volume of hybridization solution (a mixture of 2.5 ⁇ l 100% formamide, 0.5 ⁇ l 20XSSC, 0.5 ⁇ l 0.5 M phosphate (pH 7.0), 1.5 ⁇ l H 2 0 and 5 ⁇ l 20% dextran sulfate.
  • the probe solution is then denatured by heating at 75°C for 5 minutes followed by quick chilling to 4 ⁇ C.
  • the probe is further incubated for at least 2 hours at 37 ⁇ C to block out repetitive sequences before application to slides (10 ⁇ l per slide) .
  • washing solution B O.lxSSC, 60°C
  • each washing takes 5 minutes with shaking of the slide-jar. It is important that the temperature is maintained either at 45°C or 60 " C throughout the washing procedure.
  • washing solution C (4xSSC/0.05%
  • the slides are dehydrated by washing with 100 ml of 70%, 90%, and 100% ethanol.
  • Embedding solution is made by dissolving 2 gm of 1,4- Diazabicyclo-(2,2,2)-octane (DABCO) in 90 ml of glycerol for 15-30 minutes at 60 ⁇ C and 10 ml of 1.0 M Tris-HCl (pH 7.5) is added and the pH is adjusted to 8.0 with a few drops of 5 M HCl. The solution is cooled to room temperature before the addition of 100 ⁇ l of Thimerosal solution (20%) and 100 ⁇ l of propidium iodide solution (1 mg/ml) . 35 ⁇ l embedding solution is applied to each slide and covered with a coverslip before fluorescent-microscopical (Olympus) examination.
  • DABCO 1,4- Diazabicyclo-(2,2,2)-octane
  • embryos were analyzed which were derived from non-transgenic parents but injected in the pronuclear stage with hLF cDNA constructs.
  • double fluorescent spots could be detected in a number of embryos, but in the presence of a highly fluorescent background.
  • the combination of the background fluorescence and the low number of metaphase spreads per embryo does not allow unambiguous identification of the transgenic embryos.

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Abstract

Cette invention concerne des procédés fondés sur l'hybridation in situ et l'amplification enzymatique du génome qui permettent de détecter de manière précoce les transgènes intégrés dans le génome nucléaire.
PCT/US1991/004149 1991-06-12 1991-06-12 Detection precoce d'embryons transgeniques WO1992022670A1 (fr)

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PCT/US1991/004149 WO1992022670A1 (fr) 1991-06-12 1991-06-12 Detection precoce d'embryons transgeniques
AU22594/92A AU2259492A (en) 1991-06-12 1992-06-12 Early detection of transgenic emryros
PCT/US1992/005097 WO1992022647A1 (fr) 1991-06-12 1992-06-12 Detection precoce d'embryons transgeniques

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