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WO1989009272A1 - Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo - Google Patents

Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo Download PDF

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Publication number
WO1989009272A1
WO1989009272A1 PCT/US1989/001173 US8901173W WO8909272A1 WO 1989009272 A1 WO1989009272 A1 WO 1989009272A1 US 8901173 W US8901173 W US 8901173W WO 8909272 A1 WO8909272 A1 WO 8909272A1
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WIPO (PCT)
Prior art keywords
dna
cosmid
sequence
animals
mutagenesis
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PCT/US1989/001173
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English (en)
Inventor
Thomas R. Skopek
Peter K. Working
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Chemical Industry Institute Of Toxicology
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Publication of WO1989009272A1 publication Critical patent/WO1989009272A1/fr

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Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • 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 relates to use of a genetically distinct sequence to determine the mutagenic capability of chemicals.
  • the genetic sequence may be incorporated into an animal genome. After exposure of the genetically tagged animals to the chemical, the gene sequence may be retrieved from the animal and the frequency of genetic change within the sequence determined.
  • Chemical compounds or other agents which can chemically alter the DNA of a cell are capable of inducing genetic diseases such as Lesch-Nyhan syndrome, hemophilia, and sickle cell anemia.
  • many mutagens have the capability of inducing cancer in test animals.
  • a wide variety of in vitro systems have been developed using bacterial, eukaryotic, mammalian, and human cells for the detection of mutagens. These assays test for the heritable alteration of DNA by chemicals added to the cell cultures.
  • such simple systems do not accurately reflect the complex cell metabolism and drug pharmacokinetics operating in the intact animal. These factors can drastically affect the mutagenic response of an agent.
  • these systems do not provide any meaningful way of relating the induction of mutation to the induction of cancer.
  • reversion assay Techniques for studying the mutation rates of genes in bacteria commonly employ either a “reversion assay” or a “forward mutation assay”.
  • the mutation rate is studied in a gene that is active in the wild type but inactive in the bacteria to be exposed to a potential mutagen.
  • the mutation rate is monitored by scoring the specific "reverse” mutations that change the inactive gene back to the wild type configuration.
  • Such an assay cannot respond to all types of mutations, but only to those that are able to correct the original mutation that led to the inactive state.
  • the forward mutation assay In the forward mutation assay, the inactivation of a wild-type, functional target gene is detected. Such inactivation of the target gene can occur by any of the different types of mutation, including base pair substitution, frameshift mutations, deletions, and insertions. Therefore, unlike the reversion assay, it will detect all possible mutations of the target gene. Neither the reverse mutation assay nor the forward mutation assay have been used for in vivo detection of mutations of animal genes.
  • the ability to quantitate the in vivo rate of spontaneous and chemically-induced mutation in various tissues of the mouse and other animals is of great interest to genetic toxicologists.
  • knowledge of the rate of mutation produced by carcinogenic treatment regimens is valuable from a mechanistic standpoint in understanding the role of mutation in carcinogenesis.
  • the determination of the rate of mutation in the mouse may be a valuable assay in predicting the carcinogenic potential of compounds. If a sound relationship is established between the ability of a compound to induce mutation in a given tissue and its ability, to cause tumors, then a simple system testing for the ability of agents to induce mutations in the intact animal might replace at least part of the large and costly cancer studies in animals.
  • mice Techniques for inserting foreign genetic material into mice have been developed for purposes other than analysis of mutation rate. Numerous investigators have artificially introduced DNA into mice by the microinjection of fertilized mouse eggs and the subsequent transfer of the microinjected embryos into pseudopregnant host mouse mothers. See, for example, hittingham (1979). The disclosure of this reference and all other cited publications is incorporated by reference herein. Many mouse adults (up to 40%) that develop from these embryos incorporate the injected DNA, often in tandomly duplicated sequences. The mice that retain the microinjected DNA are "transgenic mice" and can both express the acquired gene and transmit it to offspring (Gordon and Ruddle, 1981). Transgenic mice have not been constructed by .the previous investigators in a manner designed to permit rescue of the inserted genetic material from the mouse and selective scoring for mutations in the genetic sequence.
  • Cosmids are plasmids carrying the cos sequence or site, a particular sequence present in the cohesive extensions at the . ends of bacteriophage lambda DNA.
  • Crude cell-free isolates from special E. coli lysogens bacteria carrying the lambda DNA in its genome
  • This technique is used extensively to package recombinant DNA-containing phage in vitro.
  • a mix of DNA is prepared, consisting of the DNA fragments to be cloned and the left and right arms of lambda.
  • the DNA fragments are ligated together, generating concatemers of lambda with the cloned DNA inserted in the middle of the monomeric lambda genomes.
  • Packaging mix (defined as the crude isolates described above) is added, thereby cutting the DNA at the cos sites and inserting it into phage particles. These then behave as normal lambda phage, which can infect and grow in E. coli.
  • This invention in its broadest aspect comprises construction of a genetically stable transgenic mouse strain for determining mutagenic damage caused in vivo by the administration of a known or suspected mutagen.
  • the invention comprises construction of a mouse strain carrying a specially engineered DNA fragment with the following characteristics:
  • the mouse is constructed by microinjection of a specially engineered DNA (vector) into a fertilized mouse egg.
  • the injected egg is returned to a recipient female and allowed to go to term.
  • the resulting transgenic mouse carries in the DNA of each of its cells several copies of the injected vector
  • the vector DNA carries a target gene (gpt) for scoring mutations, special DNA sequences permitting the in vitro rescue (isolation) of the vector from the mouse genomic
  • the vector DNA is rescued from the genomic DNA and introduced into E. coli where mutations in the target gene can be detected and quantitated.
  • the invention comprises the following steps:
  • mutant cosmid genes by isolation of DNA from the mouse tissue and packaging cosmid DNA into infectious lambda particles by in vitro packaging, followed by infection of E. coli cells with the packaged cosmid DNA and plating of the infected E. coli cells under selective conditions to detect the mutant sequences.
  • Figure 1 is a schematic drawing of a cosmid of an embodiment of the invention.
  • Figure 2 is a schematic drawing of the steps of construction of the cosmid of a preferred embodiment of the invention.
  • Figure 3 is a schematic drawing of construction of a cosmid-containing transgenic mouse.
  • Figure 4 is a schematic drawing of the mutation assay with the transgenic mouse.
  • the present invention is illustrated diagrammatically in Figures 1-4.
  • the cosmid used to infect the mouse cells is shown schematically in Figure 1 and includes: the gpt marker gene 1, a cos sequence used in in vitro packaging 2, an ampicillin resistance marker gene 3, an origin of replication from E. coli 4, and chicken DNA 5 added to make the cosmid large enough to permit packaging.
  • the steps are shown in Figure 2 and include: A) replacement of the Hindlll-BamHl fragment 6 of cosmid pHC79 with a gpt-containing Hindlll-BamHl fragment 7 from pSV2gpt;
  • strain construction includes microinjection of the cosmid DNA into the pronuclei 14 of mouse eggs 15; transfer of the 2-cell embryos 16 to pseudopregnant female mice 17 (embryos may be transferred within 2-24 hours of microinjection) ; birth and growth of the transgenic mice 18; determination of whether the cosmid DNA has been incorporated into the mice using the tail blot technique and testing of transmission of the cosmid to subsequent mouse generations by breeding.
  • the steps of the mutation assay are shown in Figure 4 and include: exposure of mice of the strain bearing the cosmid DNA fragment to a potentially mutagenic treatment 19, isolation of mouse DNA 20, in vitro packaging of mouse-derived cosmid DNA
  • E. coli colonies 23 from bacterial cells that have the ampicillin resistance marker (Minimal E Medium plus 20 ug per ml ampicillin, Richardson et al. , 1987) or (2) ampicillin and 6TG allowing growth only of E. coli cells that contain the ampicillin resistance and the mutated gpt gene markers (Minimal E Medium plus 20 ug/ml ampicillin and 8 ug/ml 6- thioguanine, Richardson et al., 1987) as the selective agents.
  • ampicillin resistance marker Minimal E Medium plus 20 ug per ml ampicillin, Richardson et al. , 1987
  • ampicillin and 6TG allowing growth only of E. coli cells that contain the ampicillin resistance and the mutated gpt gene markers (Minimal E Medium plus 20 ug/ml ampicillin and 8 ug/ml 6- thioguanine, Richardson et al., 1987) as the selective agents.
  • a forward mutation assay is used in the method of the invention allowing detection of all possible mutations of the target gene. This assay also utilizes a forward marker that is selectable on laboratory media using standard laboratory techniques.
  • the forward selection scheme employed in this invention utilizes as a forward mutation selectable marker, the bacterial gpt gene, which codes for the enzyme xanthine guanine phosphoribosyl transferase (XGPRT).
  • XGPRT xanthine guanine phosphoribosyl transferase
  • the enzyme XGPRT is an unessential enzyme that phosphoribosylates the normal purines, xanthine and guanine. It also phosphoribosylates the synthetic purine, ' 6-thioguanine (6TG), converting it from a nontoxic form to a toxic form.
  • 6TG-resistant (gpt-) E E.
  • Cosmid pHC79 described by Hohn and Collins (1980), was employed as the starting material for construction of the vector. Other cosmids that have genetic sequences with the essential properties for the invention as discussed herein may also be used.
  • Cosmid pHC79 has an E. coli origin- of replication to allow propagation of the vector of the invention in E. coli.
  • the cosmid also carries genes conferring resistance to the antibiotics ampicillin and tetracycline, providing markers to allow selection of bacteria carrying the vector.
  • the ampicillin marker alone is utilized as the bacterial selection marker, due to destruction of the tetracycline resistance gene in manipulation of the cosmid. Any other stable selective marker that can be used to select all bacteria carrying it may also be used.
  • Cosmid pHC79 also carries the cohesive site (cos site) from bacteriophage lambda.
  • a lambda phage upon infecting an E. coli host, a lambda phage first injects its 49-kilobase double-stranded genome into the bacterial cell.
  • This DNA contains an 11 base pair extension of 5'-strand at both ends (cos site).
  • the extension at the right end of the genome is complementary to the extension at the left end, so that when the two ends come together in the appropriate alignment, base pairs are formed, and the phage genome is transformed into a circular form.
  • the circular genome first undergoes bidirectional replication, generating several copies of the circular form.
  • Each circle then goes into the rolling-circle mode of replication, in which long concatemers of the phage genome are made, comprised of a plurality of copies of the genome linearly attached to each other.
  • the phage produces proteins which are able to locate the cos sequences in these concatemers, cut the DNA to regenerate the sticky 5'-ends found in the original monomeric genome, and package (insert) the monomers individually into phage protein coats.
  • the host cell is then lysed, releasing the phage particles into the medium. -15-
  • Construction of a genetically stable transgenic mouse Construction of a genetically stable transgenic mouse bearing a tandemly repeated cosmid that can be transmitted to offspring is a complex process with the following steps: (a) isolation and microinjection of the fertilized mouse embryos; (b) successful transfer of surviving embryos to suitably prepared recipient mothers; (c) detection of offspring with the tandemly repeated cosmid sequence integrated into the genome; (d) determination of the transmissibility of the microinjected DNA sequence to offspring; (e) successful rescue of the sequence from the mouse genome in vitro: and (f) production of a homogeneous strain of mice, each bearing the stably integrated sequence in sufficient quantity in all tissues.
  • the cosmid was then cut with EcoRl and ligated to EcoR ⁇ -cut chicken cell DNA in the size range of 40-50 kb to increase the size of the cosmid to that of lambda to provide efficient packaging of DNA and subsequent efficient ejection of the DNA, the efficiency of DNA packaging and ejection being a function of DNA size. Since the ligation was carried out at a high DNA concentration of 1 ug per 50 ul, long concatemers of DNA were ormed.
  • the infectious cosmid particles were used to infect E. coli cells, which were then plated on ampicillin plates using the procedure of Hohn and Collins, 1980, to select for those bacterial cells receiving the cosmid containing a gene conferring resistance to ampicillin to allow the selection.
  • a resistant colony was chosen and its cosmid DNA isolated and analyzed. As predicted, the size of the cosmid DNA was about 45 kilobases, approximately that of lambda.
  • mice Exposure of Mice to Mutagen.
  • the mice were treated with known or putative mutagen including methyl methane sulfonate, ethyl nitrosourea , mitomycin C , benzo ( alpha ) pyrene , formaldehyde , aflatoxin B ] _ , and acetylaminofluorine by administration of the putative mutagen in food or drinking w a t e r , o r by gav age , inj ec t i on ( int r amuscular , intraperitoneal , intravenous ) , inhalation or dermal absorption. Any other mutagens or mutagenesis techniques used by toxicologists may also be used .
  • mice were sacrificed and the DNA isolated from different tissues using standard techniques (Maniatis et al, 1982). The DNA was then packaged and used to infect gpt ⁇ E. coli by the technique of Hohn (1979).
  • the E. coli cells were then plated in the presence of ampicillin (20 ug/ml) to select for cells receiving the cosmid and 6TG (8 ug/ml) to select for gpt ⁇ mutants.
  • the bacteria were also plated in the presence only of ampicillin (no 6TG) to determine the total number of cells receiving the cosmid. Determination of these two bacterial counts allows calculation of the gpt " mutant fraction that exists in the packaged cosmids (Richardson et al., 1987). This determination .requires that a statistically significant number of cosmids be isolated relative to the frequency of mutations observed. If the rate of mutation in the living mouse is similar to that seen in vitro (10 ⁇ 5 ), at least 3 x 10 6 cosmids must be rescued to provide a statistically significant number of mutants for analysis.
  • the invention is preferably carried out by constructing a cosmid containing the gpt sequence; microinjecting the cosmid into a male pronucleus of a fertilized mouse egg; • producing a mouse strain from mice containing the incorporated gpt DNA; exposing mice bearing the gpt gene to selected environmental conditions; and conparing mutation rates of the gpt gene of mice exposed to the selected condition to mice not exposed to the condition.
  • Mutant cosmid genes are detected by packaging insolated mouse DNA into lambda particles followed by infection of E. coli cells with the package DNA and plating of the E. coli cells under selective conditions to detect the mutated gpt DNA.
  • the invention provides an in vivo system to study mutation rates in animals such as mice that have been exposed to potential mutagens such as particular chemicals. The induction of mutations may then be related to the induction of cancer in these animals allowing the prediction of the carcinogenic potential of the compounds. While the invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.

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Abstract

Une nouvelle lignée de souris est utilisée pour détecter des dommages mutagénétiques provoqués in vivo par l'administration d'un mutagène connu ou soupçonné. Une souris transgénique produite par microinjection d'un ADN vecteur spécialement conçu dans un ovule fertilisé de souris porte dans l'ADN de chacune de ses cellules plusieurs copies de l'ADN vecteur injecté, qui se transmet à des générations futures par le processus normal de reproduction. L'ADN vecteur porte un gène-cible de détection de mutations, des séquences spéciales d'ADN qui permettent la récupération in vitro du vecteur dans l'ADN génomique de la souris et un gène de résistance à l'ampicilline et d'origine de réplication dans E. coli qui permet de sélectionner et de multiplier le vecteur dans E. coli. Après avoir traité une souris avec un mutagène éventuel, on isole l'ADN continu dans ses différents tissus. On récupère l'ADN vecteur dans l'ADN genomique et on l'introduit dans E. coli, où l'on peut détecter et quantifier des mutations du gène-cible.
PCT/US1989/001173 1988-03-22 1989-03-21 Souris transgenique pour la mesure et la caracterisation de mutations induites in vivo WO1989009272A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0390857A4 (en) * 1987-12-15 1991-04-17 The Trustees Of Princeton University Transgenic testing systems for mutagens and carcinogens
EP0370813A3 (fr) * 1988-11-25 1991-06-19 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1992017605A1 (fr) * 1991-04-02 1992-10-15 Ingeny B.V. Procede de detection de mutations, mammifere transgenique, cellule transgenique de mammifere, et procede de recherche de proprietes mutagenes dans des agents ou des milieux
EP0671955A4 (fr) * 1992-02-14 1997-04-09 Stratagene Inc Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test.
US6063359A (en) * 1997-09-26 2000-05-16 Washington University Method for determining oncogenic activity of a substance
FR2871170A1 (fr) * 2004-06-07 2005-12-09 Proteus Sa Procede de determination de la charge mutationnelle d'une banque de genes obtenue par mutagenese aleatoire d'un gene d'interet et les moyens pour sa mise en oeuvre

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer

Patent Citations (1)

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EP0289121A2 (fr) * 1987-05-01 1988-11-02 Stratagene Test de mutagénèse utilisant des êtres non humains transgéniques porteurs des séquences d'ADN à essayer

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Title
"GENETIC TOXICOLOGY OF ENVIRONMENTAL CHEMICALS, PART B: GENETIC EFFECTS AND APPLIED MUTAGENESIS", Alan R. Liss Inc. (New York, USA), Published 1986, FEUERS et al., "The responce of kinetic parameters from selected enzymes to variant loci in congenic mice and implications for in vivo biochemical mutation tests", pages 375-382, see the entire document. *
CANADIAN JOURNAL OF GENETICS AND CYTOLOGY, (Ottowa, Canada), Volume 28, issued 1986, SINGH et al., "Genotype-and age-associated in vivo Cytogenic alterations following mutagenic exposures in mice", pages 286-293, see the entire document. *
FASEB JOURNAL, (Bethesda, USA), Volume 2(6), issued 25 March 1988, SHORT et al.: "A transgenic model for the identification of genetic lesions", abstract 8515. *
MUTATION RESEARCH, Elsevier (Amsterdam, Netherland), Volume 181, issued 1987, LOHMAN et al.: "DNA methods for detecting and Analyzing mutations in vivo", pages 227-234, see pages 228-229 in particular. *
NUCLEIC ACIDS RESEARCH, (London, UK), Volume 10, Number 4, issued 1982, LINDENMAIER et al., "Gene shuttling: moving of cloned DNA into and out eukaryotic cell", pages 1243-1256, see the entire document. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA (Washington, USA), Volume 80, issued September 1983, LAU et al., "Versatile cosimid vectors for the isolation, expression and rescue of gene sequences: studies with the human alpha-globin gene cluster", pages 5225-5229, see the entire document. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, USA (Washington, USA), Volume 83, issued February 1986, GLAZER et al., "Detection and analysis of UV-induced mutations in mammalian cell DNA using a lambda page shuttle vector", pages 1041-1044, see the entire document. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0390857A4 (en) * 1987-12-15 1991-04-17 The Trustees Of Princeton University Transgenic testing systems for mutagens and carcinogens
EP0370813A3 (fr) * 1988-11-25 1991-06-19 Exemplar Corporation Dosage rapide par criblage de mutagénèse et tératogénèse
WO1992017605A1 (fr) * 1991-04-02 1992-10-15 Ingeny B.V. Procede de detection de mutations, mammifere transgenique, cellule transgenique de mammifere, et procede de recherche de proprietes mutagenes dans des agents ou des milieux
EP0671955A4 (fr) * 1992-02-14 1997-04-09 Stratagene Inc Test de mutagenese utilisant des animaux non humains transgeniques portant des sequences d'adn test.
US6063359A (en) * 1997-09-26 2000-05-16 Washington University Method for determining oncogenic activity of a substance
US6428969B1 (en) 1997-09-26 2002-08-06 Ludwig Institute For Cancer Research Method for determining oncogenic activity of a substance
FR2871170A1 (fr) * 2004-06-07 2005-12-09 Proteus Sa Procede de determination de la charge mutationnelle d'une banque de genes obtenue par mutagenese aleatoire d'un gene d'interet et les moyens pour sa mise en oeuvre
WO2006003298A3 (fr) * 2004-06-07 2006-05-26 Proteus Procede de determination de la charge mutationnelle d'une banque de genes obtenue par mutagenese aleatoire d'un gene d'interet et les moyens pour sa mise en oeuvre

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