WO1992021229A1 - Mammiferes transgeniques ζx174 - Google Patents

Mammiferes transgeniques ζx174 Download PDF

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Publication number
WO1992021229A1
WO1992021229A1 PCT/US1992/004593 US9204593W WO9221229A1 WO 1992021229 A1 WO1992021229 A1 WO 1992021229A1 US 9204593 W US9204593 W US 9204593W WO 9221229 A1 WO9221229 A1 WO 9221229A1
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mammal
dna
vector
phage
cells
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PCT/US1992/004593
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James G. Burkhart
Heinrich V. Malling
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The United States Of America, Represented By The Secretary, Department Of Health And Human Services
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Publication of WO1992021229A1 publication Critical patent/WO1992021229A1/fr

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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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

  • the present invention relates to a transgenic non-human mammal whose chromosomal genome is stably integrated with double-stranded DNA viral vector ⁇ X174.
  • the present invention also relates to methods of detection of in vivo mutation.
  • the present invention provides a novel vehicle for the detection and study of in vivo mutation directly at the DNA level in somatic and germinal tissue using simple techniques and very few animals.
  • the approach has been to utilize a small DNA viral vector, ⁇ X174, stably integrated into the chromosomal genome of transgenic mice. There is no expression or selection of the vector's mutant or normal phenotype in the host cells or tissue.
  • the viral vector can be recovered from the DNA of the host, and mutations within genes of the vector can be detected and analyzed.
  • the use of the same vector can be expanded to a variety of eukaroytes and prokaryotes.
  • TNO Medical Biological Laboratory (Rijswijk, the Netherlands) has recently developed a transgenic mouse system for the study of mutation based on incorporation of lambda phage containing the lacZ gene. These mice are being marketed through Hazelton (Kensington, Maryland) in the United States. However, there are some fundamental differences between approaches using lambda-based vectors and the use of transgenic
  • linear 0X174 containing the am3 mutation was cotransfected into TK mouse L cells to form a cell line containing 5 potentially recoverable ⁇ X copies stably integrated in a single array into the chromosomal genome.
  • the inserted ⁇ X DNA was shown to be unmethylated.
  • methods were developed to recover to ⁇ X phage from the host genome. Development of methods at this stage included partial purification of the ⁇ x sequence by digestion of the chromosomal DNA with Sau3A I, Pvu II, and Pst I followed by S-1000 column chromatography (Sau3A I and Pvu II cleave the host
  • the ⁇ X transgenic cell line was treated with 20mM ethylmethane sulfonate (an alkylating agent) , and it was determined that mutations could be detected as reversions of am3 among recovered phage. Overall, these previous experiments demonstrated that ⁇ X could be stably integrated into a host mammalian cell genome and efficiently recovered and that mutations could be detected among phage recovered from treated cells. (Burkhart et al. Mutation Research. 213:125 (1989).)
  • ⁇ X174 which vector is introduced into the mammal, or an ancestor of the mammal, at an embryonic stage.
  • step (ii) isolating genomic DNA from the mammal (i ⁇ ) partially purifying ⁇ X174 DNA from the isolated genomic DNA of step (ii) ;
  • step (iv) transfecting by electroporation bacterial cells with the partially purified ⁇ X DNA of step (iii) and replicating ⁇ X phage, which bacterial cells are selected for resistance to ⁇ X infection,* and
  • step (v) growing replicated ⁇ X phage of step (iv) on selective and non-selective bacterial host strains to identify mutations. It is a further object of the present invention to provide a method for detecting mutations at the DNA level of a non-human mammal, using the above-described method, where the bacterial cells of step (iv) are developed from E__ _______. and have the characteristics of being resistant to infection by ⁇ X phage and exhibiting high transfection efficiency for ⁇ X by electroporation.
  • Another object of the present invention is to provide a method for detecting mutations at the DNA level of a non-human mammal, using the above-described method, where the mutations are detected by specific reversions from a mutant to normal phenotype.
  • Another object of the present invention is to provide a method for detecting mutations at the DNA level of a non-human mammal, using the above-described method, where step (v) further comprises deriving the total number of rescued progeny ⁇ X174 phage and the number of mutants.
  • FIGURES Figure 1 ⁇ X Copy Number in Founder Animal #37. Pst 1 digestion of genomic DNA from a founder, animal (#37) followed by electrophoresis with prepared standards of mouse DNA containing ⁇ X RFDNA in known concentrations indicates that approximately 50 copies had been integrated into the mouse genomic DNA. Of the first 4 founders animals, all had approximately the same number of copies.
  • Figure 2 ⁇ X Copies on Homozygous 54 Line Mice. Pst 1 digestion of DNA from subsequent breeding of founder 54 indicates that the line is homozygous for the vector and that there are about 100 copies of the ⁇ X sequence incorporated into the mouse genome.
  • Figure 3 ⁇ 6X Line 54. Transmission and litter data from line 54 mice indicates that the vector does not appear to have any detrimental or recessive lethal effects. (The normal size for a C57BI6/J litter is 6.5.)
  • Figure 4 Methylation of ⁇ X174 in Transgenic Mouse Line 54. Digestion of ⁇ X RFDNA and genomic DNA from transgenic mice with Hpa II (methylation sensitive) and Msp (methylation insensitive) indicates that the vector DNA is methylated in the host genome.
  • Figure 5 Recovery of ⁇ X174 from a Transgenic Mouse with Approximately 50 Potentially Active Copies.
  • Example of phage recovery from the mouse genome demonstrates sufficient numbers to allow mutation analysis.
  • Figure 6 am3 Reversions in Chromosomallv-Integrated ⁇ X Induced by N-Ethyl N- Nitrosourea in Mouse Liver. Analysis of mutations at am3 in phage recovered from untreated mice, and mice treated with 200 mg/kg n-ethyl-n-nitrosourea indicates a 5-fold increase in mutation induction in somatic tissues of treated animals.
  • the present invention relates to a transgenic non-human mammal (for instance, a mouse) all of whose cells, including germinal and somatic cells, contain a stably integrated DNA viral vector ⁇ X174.
  • the present invention also relates to the stable integration of DNA vector ⁇ X174 into the genome of an ancestor of the non-human mammal.
  • the vector is introduced into the mammal at an embryonic stage, by methods well-known in the art.
  • the Applicants Using a shuttle-vector approach, the Applicants have developed a method for the study of mammalian mutagenesis by devising a small vector (5.3 kb in length) that has been stably integrated into the chromosomal genome of mammals (i.e. transgenic mice) with no expression of ⁇ X in the host and no detectable homology between the ⁇ X sequence and the mammalian genome. Further, there was no selection of vector normal or mutant phenotype in the host cells or tissue.
  • viable phage can be efficiently recovered from the chromosomal DNA of the host mammal, and mutations can be detected among phage recovered from treated cells. Mutations are not significantly induced by the recovery process. Additionally, there is an increase in the mutation frequency of the vector after treatment of the host with a known mutagen.
  • the present invention relates to a method of detecting mutations at the DNA level in germinal and somatic cells of non-human mammals (for instance, mice) comprising the steps of:
  • step (iii) partially purifying ⁇ X174 DNA from the isolated genomic DNA of step (ii) ;
  • step (iv) transfecting, advantageously by electroporation, bacterial cells with the partially purified ⁇ X DNA of step (iii) and replicating ⁇ X phage, which bacterial cells are selected for resistance to ⁇ X infection;
  • step (v) growing replicated phage of step (iv) on selective and non-selective bacterial host strains to identify mutations.
  • the vector is a shuttle vector, and mutations are detected by specific reversions from a mutant to normal phenotype.
  • the marker gene of step (i) may be of the group comprising am3, lad, aml6, aml8, strp resistance and sup+ F.
  • the bacterial cells of step (iv) may be taken from a strain developed from E. coli C. (designated C ⁇ XRl) , and act effectively as a recovery mechanism of ⁇ X. These cells have been developed to be resistant to infection by ⁇ X phage particle and exhibit high transfection efficiency for ⁇ X by electroporation. Hence, there is little or no chance for the recovery of ⁇ X DNA sequences from host genomic DNA to be contaminated. For instance, contamination could occur by wild type ⁇ X, which would negate the accuracy of any mutation detection data. Likewise, there is little or no chance for reinfection by ⁇ X from accidental lysis of bacterial cells. The recovery E.
  • coli strain C ⁇ XRl does not contain restriction enzymes that cleave DNA that has been CpG methylated, unlike immortal mammalian cell lines.
  • ⁇ X in transgenic cell lines is not methylated, whereas ⁇ X in the transgenic mammals of the present invention is methylated.
  • Deposit is not intended to limit the concept of the present invention to the particular biological material deposited. The deposit will be maintained according to applicable laws, and will be available to the public as required.
  • the bacterial host strain of step (v) may be E. coli.
  • step (v) may further comprise deriving the total number of rescued progeny ⁇ X174 phage and the number of mutants.
  • the present invention demonstrates a number of significant advantages over the prior art use of lambda-based vectors.
  • the integrated lambda cloning vector is approximately 50 kb in length, whereas ⁇ X is 5 kb.
  • the small size of ⁇ X means that many more copies can be inserted into the host genome with less overall potential for genomic disruption or reduction in viability.
  • the smaller size of the ⁇ X vector also means that there is less likelihood for cleavage (or rearrangement) of the vector sequence during recovery from the host or in any subsequent analysis.
  • lambda-based vectors as a standard laboratory cloning vehicle demonstrates that in use as mutation shuttle vectors, they may be subject to internal rearrangement independent of actual mutation events in the host. Size-related fragmentation and rearrangement would cause misrepresentation of mutations occurring in the host organism.
  • mutation detection is superior in the present invention. Mutations are currently being detected in ⁇ shuttle vectors as forward events. In ⁇ X174 vectors, mutations are detected as specific reversions. Forward mutation detection in lambda means that the target sequence is large (in the range of 3000bp) . But in order to be detected, the mutation must inactivate the translation product. All viable lambda must be screened histochemically and the nature of any putative mutation can only be determined by sequence analysis. Although the large target size associated with forward mutation detection is somewhat desireable because of the potential number of mutations, the indicator inactivation is not specific within the target gene. Furthermore, forward mutation detection tends to have a high background and can be biased towards mutations producing internal stop codons, frame shifts, or sequence rearrangement. This high background makes analysis of germinal mutations and low- frequency somatic mutations very difficult.
  • mutant detection method used in the ⁇ X174 transgenic system also allows the investigator to distinguish mutations that occurred in the host from those that arose as de novo events after excision of the vector from the host. Background and spontaneous mutations are very low, and certain events not usually observable in a forward mutation system (such as transitions at an A:T pair) can be seen.
  • ENU N-ethyl-N-nitrosourea
  • the ⁇ X174 vector system facilitates better recovery of the phage DNA.
  • Lambda phage are recovered from host DNA by electrophoretic purification of the phage sequence followed by packaging of the phage DNA into virions using highly specialized cell extracts. These cell extracts are difficult to produce and, when purchased from a vendor, may constitute a considerable expense for any laboratory using the lambda vector.
  • 0X174 can be easily partially purified from the host organism by restriction enzyme digestion of the host genomic DNA and a simple modification of currently available mini-column technology, without any requirement for electrophoresis or probe hybridization to identify the specific sequence. The phage can then be recovered by electroporation into an E.
  • coli strain developed to be resistance to infection by the ⁇ X phage particle and to exhibit high transfection efficiency for ⁇ X by electroporation.
  • the competency of the eletroporation protocol is in the range of 10- 20%.
  • Recovery of lambda and ⁇ X174 are comparable per genomic copy.
  • the ⁇ X174 system is also advantageous in the production of the transgenic strains.
  • the commercially available transgenic strains carrying lambda vectors have been produced by injection of Fl hybrid ova in order to take advantage of enhanced vigor of the eggs produced in such crosses.
  • transgenic insert can be made homozygous in a particular line, all other genes that differ in the two parental types will assort independently; thus, a parental type homozygous for the insert cannot be reproduced; 2) each line produced by brother-sister matings of the Fl generation from the transgenic founder will constitute a recombinant inbred line; 3) data from whole animal studies using biological markers in response to toxicological exposure indicates that such lines can be very different in response both from one another and from the parent strains due to segregation of genes important in the processes of xenobiotic metabolism.
  • inbred and independent mutation markers in the method of the present invention means that single lines can be constructed with multiple specific markers, and a forward ⁇ X mutation marker can also be developed. The skilled artisan would then be able to distinguish between various types of induced mutation events in a single mouse without the a priori requirement for sequence analysis of each mutant.
  • Example 1 Production of Transgenic Mice ⁇ X174 shuttle vector is constructed using methods well known in the art, according to figure 8.
  • Purified ⁇ X RFDNA is linearized by digestion with Pst I, then chloroform-phenol extracted and ethanol precipitated.
  • the linear ⁇ X DNA is ligated at various short (i.e. 30 sec-5 min) intervals with T4 DNA ligase or E. coli DNA ligase.
  • Aliquots of the ligated DNAs are examined by agarose gel electrophoresis and ethidiu bromide staining; a sample is selected based on the existence of a ladder of catenated ⁇ X copies between 2 and 10.
  • the fertilized mouse ova are then injected with 100-250 copies of the ⁇ X RFDNA according to standard procedures available in Hogan et al, "Manipulating the Mouse Embryo" Cold Spring Harbor, (1986) .
  • Genomic DNA from tail clips of mice resulting from the injected ova are screened for the presence of ⁇ X sequences after Pst I digestion. For any mouse containing the sequence, the number of copies and insertion sites are estimated (see enclosed figures) .
  • the transgenic founders are mated and the progeny similarly screened. Transgenic males and females from the same founder are subsequently mated to produce a line of mice homozygous for the particular ⁇ X insertion.
  • tissue may be excised and used immediately or frozen at -70"C as needed.
  • Approximately 1 gm tissue (or less as applicable) is homogenized on ice in 20mL cold 0.25M sucrose- TE (10M Tris, ImM ETA, pH 7.6) using a polytron (Brinkman or equivalent) .
  • the homogenate is layered over 20ml cold 0.35M sucrose-TE and centrifuged for 10 min at 4'C and 3000rpm in a standard preparative centrifuge (IEC or equivalent) equipped with a swinging bucket rotor.
  • the suspension is incubated ta 55 * C for 2hr. with gentle mixing, then extracted twice with an equal volume chloroform-phenol (1:), twice with chloroform, then ethanol precipitated.
  • the DNA is suspended in TE at a final concentration in the range of l ⁇ g/ ⁇ L.
  • lOO ⁇ g host DNA is digested simultaneously with 100U each PST I, PVU II, and SAU3A 1 at 37*C for 3hr with gentle mixing.
  • the restriction enzymes are removed by proteinase K digestion (19 ⁇ g) at 55"C for 1 hr followed by extraction nd precipitation as described.
  • the pellet is resuspended in approximately 25-50 ⁇ L TE and warmed to insure that the DNA is in a homogenous solution.
  • the DNA is loaded onto the minicolumn and eluted with lmL TE.
  • the eluent is made compatible for T4 DNA ligase assay conditions (not containing PEG) ; 4-10U ligase is added and the solution is incubated at approximately 12 "C for 2-3 hr. The ligation is stopped by heating to 65*C for 10 min and exchanging the solution 3 times against sterile filtered after using 300,000 nmwl miniature filters and centrifugation. The final volume reduction is suspended in 500 ⁇ l water. We are constantly making improvements in this area, and there is a possibility for removing the S- 1000 column step and still maintaining recovery.
  • Cells for electroporation are prepared in advance and stored according to this general protocol.
  • the particular culture for electroporation of recovered ⁇ X174 ds DNA has been developed from E.coli C by selection for resistance to ⁇ X infection and as a fast growing clone in NZY medium.
  • a log culture can be prepared, and DMSO added to a final concentration of 7%.
  • the culture is aliquoted and frozen at - 70'C until use.
  • the cultures for preparation of competent cells are started by inoculating 50mL of NZY medium with a very small amount of, cells from the frozen stock culture. This culture is grown overnight with vigorous shaking.
  • a second overnight culture is then prepared from this culture by inoculating the same amount of medium with a very small amount (lOO ⁇ L) of the first overnight culture.
  • the concentration of the cells is adjusted to 2.5 x 10 10 cells per ml; the cell suspension is aliquoted into eppendorf tubes, quickly frozen in liquid nitrogen and stored at -70*C.
  • the am3 reversions is determined by plating the phage suspension on E. coli C. That baterial strain does not contain any suppressor and is therefore selective for am3 reversions. The plating cultures are prepares as the CO-2 culture.
  • 2 culture is checked for its infectivity and possible contamination with spurious phages.
  • the infectivity check is done by plating 20 ⁇ l of a phage suspension which contain 40-80 infective centres. It is practical to make many individual stock cultures of ⁇ X174 am3 cs70 in borate is stable in the coldroom at 4'C for several years.
  • the presence of spurious phages in the bacteria culture is done by plating 4 x 1 ml of the bacteria culture as described above but without any addition of phages.
  • the E. coli C culture is checked for infectivity and contamination with spurious phages.
  • the check for infectivity is done by using the ⁇ X174 wild type phage.
  • the cs70 mutant serves as a check on the genotype of the revertants detected on the plates from the DNA from the animals.
  • the wildtype does not contain the cs70 mutant.
  • the infectivity is checked by adding .5 ml of a phage suspension which contain approximately 50-100 infective centres following the procedure for plating a determine the reveres mutation frequency.
  • Plating for the check of presence of spurious phages is done by plating 4 x 10 ml bacteria solution without addition pf phages.
  • the total number of phages in the sample equal:
  • 15 is the total volume of the electroporesis culture. ⁇ l plated is usually 20 ⁇ l.
  • the competence of the cells can in general be described as the efficiency by which the ⁇ X174 DNA has entered the bacterial cells.
  • the competence is proportional to the concentration of the DNA within many orders of magnitude. Nevertheless we have selected a concentration of ⁇ X174 RF DNA
  • Competence (double standard) which yield the same number of phages as can be expected from a good DNA sample from the animals. Competence equal:
  • the burst size of the ⁇ X174 is approximately 100.
  • the total number of recovered progeny from the mammalian is therefore equal to the total number of phages in the sample divided with the burst size.

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Abstract

La présente invention se rapporte à un mammifère transgénique non-humain tel qu'une souris, dont toutes les cellules germinales et somatiques contiennent un vecteur navette viral d'ADN à intégration stable, ΖX174, ce vecteur étant introduit dans ledit mammifère, ou un ancêtre dudit mammifère, à un stade embryonnaire. L'invention se rapporte aussi à des procédés de détection de mutations au niveau de l'ADN dans des cellules germinales et somatiques de mammifères non humains, ces mutations étant détectées par des réversions spécifiques d'un phénotype mutant à un phénotype normal.
PCT/US1992/004593 1991-06-07 1992-06-05 Mammiferes transgeniques ζx174 WO1992021229A1 (fr)

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US710,428 1991-06-07

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022622A1 (fr) * 1996-11-18 1998-05-28 Wisconsin Alumni Research Foundation Procede d'identification de mutants et de molecules
WO1999001577A1 (fr) * 1997-07-02 1999-01-14 Hexagen Technology Limited Echantillons de criblage de reproduction et genetique d'animaux mutagenises
US6949692B2 (en) 1996-11-18 2005-09-27 Wisconsin Alumni Research Foundation Method for identifying mutants and molecules

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)

* 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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MUTATION RESEARCH, Volume 213, issued 1989, J.G. BURKHART et al., "Mutagenesis of phi X174 am3 cs70 Incorporated into the Genome of Mouse L-Cells", pages 125-134. *
NATURE, Vol. 265, issued 24 February 1977, SURGER et al., "Nucleotide sequence of Bacteriophage...DNA", pages 687-688. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022622A1 (fr) * 1996-11-18 1998-05-28 Wisconsin Alumni Research Foundation Procede d'identification de mutants et de molecules
US6949692B2 (en) 1996-11-18 2005-09-27 Wisconsin Alumni Research Foundation Method for identifying mutants and molecules
WO1999001577A1 (fr) * 1997-07-02 1999-01-14 Hexagen Technology Limited Echantillons de criblage de reproduction et genetique d'animaux mutagenises
AU742368B2 (en) * 1997-07-02 2002-01-03 Hexagen Technology Limited Reproductive and genetic screening samples of mutagenised animals

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