WO1998024307A1 - Procede servant a obtenir des animaux modifies genetiquement - Google Patents

Procede servant a obtenir des animaux modifies genetiquement Download PDF

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Publication number
WO1998024307A1
WO1998024307A1 PCT/AU1997/000817 AU9700817W WO9824307A1 WO 1998024307 A1 WO1998024307 A1 WO 1998024307A1 AU 9700817 W AU9700817 W AU 9700817W WO 9824307 A1 WO9824307 A1 WO 9824307A1
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blastocysts
embryos
animal
mouse
cells
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Heinrich Korner
Frances Anne Lemckert
Jonathan Douglas Sedgwick
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Centenary Institute of Cancer Medicine and Cell Biology
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Centenary Institute of Cancer Medicine and Cell Biology
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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
    • 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

  • the present invention relates generally to genetically modified animals and to a method of producing same. More particularly, the present invention provides a method of producing genetically modified animals by introducing embryonic stem cells into blastocysts which have been isolated at an early embryonic stage and then cultured to fully expanded blastocyst stage in vitro.
  • the method of the present invention is particularly useful for obtaining blastocysts from animals exhibiting delayed embryonic development and/or from animals which generally yield low numbers of mature blastocysts after superovulation procedures or following natural mating.
  • the subject method also permits the generation of a range of genetically modified animals. These animals are particularly useful in assessing gene function and gene expression and their effects on the modified animal's phenotype.
  • ES cells are the pluripotent out growths of the inner cell mass of blastocysts and can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute normally to all tissues including the germline (1).
  • ES cells are injected into blastocysts from a selected animal strain which are then implanted into a pseudopregnant female animal for development.
  • mice and other animals with a pure genetic background is highly desirable since back-crossing potentially causes conflicting and inconsistent results (2-4).
  • ES cells from a white mouse eg. 129 are injected into blastocysts derived from a black strain and implanted into a suitable pseudopregnant mouse. Chimeric pups are produced and these are bred to produce homozygous knockout mice on the 129 strain background.
  • mouse strain 129 can be difficult to breed and thus is usually crossed to other strains. Concerns have been raised about the ambiguity of results derived from back-crossed animals (4).
  • BALB/c strain blastocysts are the preferred recipient (5).
  • a major disadvantage of using blastocysts from the BALB/c strain is the delayed embryonic development exhibited by this strain.
  • BALB/c mice are also poorly susceptible to superovulation procedures.
  • One aspect of the present invention contemplates a method for producing a chimeric animal, said method comprising isolating embryos from an animal at an early embryonic stage, culturing said embryos in vitro to an injectable blastocyst stage, introducing ES cells from another animal to said cultured blastocysts and then implanting the injected blastocysts into a suitable animal for development of chimeric off-spring.
  • the present invention provides a method for producing a chimeric animal capable of germ-line transmission of an introduced genetic trait said method comprising isolating embryos from an animal at an early embryonic stage, culturing said embryos in vitro to an injectable blastocyst stage, introducing ES cells from another animal into said cultured blastocysts, implanting the injected blastocysts into a suitable animal for development of chimeric off-spring and then back-crossing a chimeric animal off-spring with the same animal species from which the ES cells were isolated or an FI hybrid thereof and selecting off-spring exhibiting said introduced genetic trait.
  • Another aspect of the present invention contemplates a method for producing a chimeric mouse said method comprising introducing ES cells from one mouse into blastocysts from another mouse said blastocysts obtained from embryos isolated at an early embryonic stage and cultured to maturity in vitro and then implanting said blastocysts into a suitable mouse for development of chimeric mice off-spring.
  • the present invention relates to a method for producing a chimeric mouse capable of germ-line transmission of an introduced genetic trait, said method comprising introducing ES cells from one mouse into blastocysts from another mouse said blastocysts prepared from embryos obtained at an early embryonic stage and cultured to mature blastocysts in vitro, implanting said blastocysts into a suitable mouse for development of chimeric mice offspring and then back-crossing a chimeric mouse off-spring with the same species of mouse from which the ES cells were isolated or an FI hybrid thereof and selecting off-spring exhibiting said introduced genetic trait.
  • Yet another aspect of the present invention provides a method for producing a genetically modified animal, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant animal for development of genetically modified off-spring.
  • the present invention is directed to a method for producing a genetically modified animal capable of germ-line transmission of a genetic modification, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, ES cells carrying a modified genomic sequence, implanting the blastocysts into a suitable implant animal for development of genetically modified off-spring and then back-crossing a genetically modified off-spring with the same species of animal from which the ES cells were isolated or an FI hybrid thereof and selecting an off-spring exhibiting said genetic modification.
  • Yet another aspect of the present invention provides a method for producing a genetically modified mouse, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant mouse for development of genetically modified off-spring.
  • the present invention is directed to a method for producing a genetically modified mouse, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence, implanting the blastocysts into a suitable implant mouse for development of genetically modified offspring and then back-crossing a genetically modified off-spring with the same species of mouse from which the ES cells were isolated or an FI hybrid thereof and selecting an off-spring exhibiting said genetic modification.
  • Still yet another aspect of the present invention provides a genetically modified animal produced by isolating animal embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, animal ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant animal for development of genetically modified offspring.
  • the present invention relates to a genetically modified animal produced by isolating animal embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, animal ES cells carrying a modified genomic sequence, implanting the blastocysts into a suitable implant animal for development of genetically modified offspring and then back-crossing a genetically modified off-spring with the same species of animal from which the ES cells were isolated or an FI hybrid thereof and then selecting an off-spring exhibiting said modification.
  • Yet a further aspect of the present invention provides a genetically modified mouse produced by isolating mouse embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, mouse ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant mouse for development of genetically modified offspring.
  • Yet a further aspect of the present invention provides a genetically modified mouse produced by isolating mouse embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, mouse ES cells carrying a modified genomic sequence, then implanting the blastocysts into a suitable implant mouse for development of genetically modified offspring and then back-crossing a genetically modified off-spring with the same species of mouse from which the ES cells were isolated or an FI hybrid thereof and then selecting an off-spring exhibiting said genetic modification.
  • Still a further aspect of the present invention provides a genetically modified mouse produced by isolating embryos from BALB/c or related mice at an early embryonic stage, culturing said embryos in vitro until maturation to blastocyst stage introducing into said blastocysts, ES cells isolated from C57BL 6 or related mice wherein said ES cells carry a modified genomic sequence and then implanting said blastocysts into an implant mouse such as but not limited to C57BL/6 or an FI hybrid thereof and then selecting an off-spring exhibiting said genetic modification.
  • the present invention is related to a genetically modified mouse produced by isolating embryos from BALB/c or related mice at an early embryonic stage, culturing said embryos in vitro until maturation of said blastocysts, introducing into said blastocysts, ES cells isolated from C57BL/6 or related mice wherein said ES cells are genetically modified by insertion, deletion and/or substitution of a genetic sequence and then implanting said blastocysts into an implant mouse such as but not limited to C57BL 6 or an FI hybrid thereof and then back- crossing a chimeric mouse produced by said method with a C57BL/6 mouse or a C57BL/6 x BALB/c FI hybrid mouse and selecting an off-spring exhibiting said genetic modification.
  • an implant mouse such as but not limited to C57BL 6 or an FI hybrid thereof and then back- crossing a chimeric mouse produced by said method with a C57BL/6 mouse or a C57BL/6 x BALB/c FI hybrid mouse and selecting an
  • Another aspect of the present invention contemplates the method for isolating embryos from animals exhibiting delayed embryonic development or from animals which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, said embryos useful in the production of chimeric animals, said method comprising isolating said embryos from an animal at an early embryonic stage and then culturing said embryos in vitro until maturation into blastocysts.
  • the present invention contemplates the method for isolating embryos from mice exhibiting delayed embryonic development or from mice which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, said embryos useful in the production of chimeric mice, said method comprising isolating said embryos from a mouse at an early embryonic stage and then culturing said embryos in vitro until maturation into blastocysts.
  • the present invention is directed to a method for isolating embryos from BALB/c mice or other mice exhibiting delayed embryonic development or from mice which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, said embryos useful in the production of chimeric mice, said method comprising isolating said embryos from a mouse at an early embryonic stage and then culturing said embryos in vitro until maturation into blastocysts.
  • the same species of animal is in fact the same strain of animal.
  • the present invention is directed to genetically modified mice and a method of producing same.
  • the subject invention provides methodology to permit the generation of high yields of blastocysts from animals exhibiting delayed embryonic development and/or from animals which generally yield low numbers of mature blastocysts after superovulation procedures or following natural mating.
  • ES cells carrying genetic modifications to their genomes can then be introduced into the blastocysts for introduction into a suitable implant animal.
  • one aspect of the present invention contemplates a method for producing a chimeric animal, said method comprising isolating embryos from an animal at an early embryonic stage, culturing said embryos in vitro to an injectable blastocyst stage, introducing ES cells from another animal into said cultured blastocysts and then implanting the injected blastocysts into a suitable animal for development of chimeric off-spring.
  • the preferred chimeric animals contain a genetic trait capable of transmission through their germ-line.
  • the resulting chimeric off-spring can be back- crossed, generally with the same species and, more preferably, the same strain of animal from which the ES cells are isolated or an FI hybrid thereof and selecting off-spring which exhibit the genetic trait.
  • another aspect of the present invention provides a method for producing a chimeric animal capable of germ-line transmission of an introduced genetic trait said method comprising isolating embryos from an animal at an early embryonic stage, culturing said embryos in vitro to an injectable blastocyst stage, introducing ES cells from another animal into said cultured blastocysts and then implanting the injected blastocysts into a suitable animal for development of chimeric off-spring and then back-crossing a chimeric animal off-spring with the same animal species, or more preferably same animal strain, from which the ES cells were isolated or an FI hybrid thereof and selecting off-spring exhibiting said introduced genetic trait.
  • the system When the species of animal from which the blastocysts and ES cells are obtained is the same species as the implant animal, then the system is referred to as a "homologous" system. When the species of animal is different, then the system is referred to as “heterologous”system.
  • An example of a heterologous system is the use of ovine embryos in goat implant animals. Homologous systems are the preferred systems.
  • the implant animal can be considered as an "incubator" animal and is generally a pseudopregnant female. Conveniently, the implant animal may also be an FI hybrid. With respect to mice, for example, a suitable implant animal is a C57BL/6 mouse or the FI hybrid C57BL/6 x BALB/c mouse.
  • Chimeric animals are conveniently detected by skin or fur colour.
  • ES cells from black mice eg. C57BL/6
  • blastocysts from white mice eg. BALB/c
  • chimeric animals are those which are black and white.
  • the present invention extends, however, to a range of other means for detecting chimeras such as but not limited to eye colour, genetic screening (eg. by PCR, dot blot, etc.), fur length or other traits such as tail length or a combination of phenotypes.
  • Skin or fur colour is, however, up to the present time, the most convenient indication of chimerism.
  • the ES cells carry a modified genomic sequence when injected into the blastocysts.
  • Modifications to a genomic sequence include for example, inserting, deleting and/or substituting a nucleotide sequence.
  • the modification may be, the introduction of an antisense molecule, a sense molecule for co-suppression, a nucleotide sequence encoding a particular protein or a molecule capable of replacing an existing endogenous sequence or introducing a new nucleotide sequence by homologous recombination.
  • the genetic modification may entail a recombinase system such as but not limited to the cxc/lox (15, 16) and the flp//rt systems (17, 18).
  • Such systems generally involve loci (eg. lox or frt) for DNA recombination flanking a selected nucleotide sequence in combination with a recombinase (eg. ere or flp).
  • a recombinase eg. ere or flp.
  • the recombinase specifically recognises the recombinant loci resulting in site-specific recombination and deletion of the nucleotide sequence.
  • the genetically modified animals obtained via these ES cells are particularly useful models for assessing gene expression and function and their effects on phenotype.
  • another aspect of the present invention contemplates a method for producing a genetically modified animal, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant animal for development of genetically modified off-spring.
  • the present invention is directed to a method for producing a genetically modified animal capable of germ-line transmission of a genetic modification, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, ES cells carrying a modified genomic sequence implanting the blastocysts into a suitable implant animal for development of genetically modified off-spring and then back-crossing a genetically modified off-spring with the same species of animal, or more preferably the same strain of animal, from which the ES cells were isolated or an FI hybrid thereof and selecting an off-spring exhibiting said genetic modification.
  • mice The present invention is hereinafter described with reference to the production of genetically modified mice. However, this is done with the understanding that the present invention is applicable to ES cells and blastocysts from all animals including but not limited to humans, primates, livestock animals (eg. sheep, pigs, cows, horses, donkeys, goats), laboratory test animals (eg. rabbits, guinea pigs, rats) companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer).
  • livestock animals eg. sheep, pigs, cows, horses, donkeys, goats
  • laboratory test animals eg. rabbits, guinea pigs, rats
  • companion animals eg. dogs, cats
  • captive wild animals eg. foxes, kangaroos, deer.
  • another aspect of the present invention contemplates a method for producing a chimeric mouse said method comprising introducing ES cells from one mouse into blastocysts from another mouse said blastocysts obtained from embryos isolated at an early embryonic stage and cultured to maturity in vitro, and then implanting said blastocysts into a suitable mouse for development of chimeric mice off-spring.
  • the present invention provides a method for producing a chimeric mouse capable of germ-line transmission of an introduced genetic trait said method comprising isolating embryos from a mouse at an early embryonic stage, culturing said embryos in vitro to an injectable blastocyst stage, introducing ES cells from another mouse to said cultured blastocysts, implanting the injected blastocysts into a suitable mouse for development of chimeric off-spring and then back-crossing a chimeric mouse off-spring with the same mouse species, or more preferably the same mouse strain, from which the ES cells were isolated or an FI hybrid thereof and selecting off-spring exhibiting said introduced genetic trait.
  • Yet another aspect of the present invention provides a method for producing a genetically modified mouse, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant mouse for development of genetically modified off-spring.
  • the present invention is directed to a method for producing a genetically modified mouse, said method comprising isolating embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts, and introducing into said blastocysts, ES cells carrying a modified genomic sequence, implanting the blastocysts into a suitable implant mouse for development of genetically modified off-spring and then back-crossing a genetically modified off-spring with the same species, of more preferably strain, of mouse from which the ES cells were isolated or an FI hybrid thereof and selecting an off-spring exhibiting said genetic modification.
  • the present invention is particularly useful for establishing blastocysts from animals exhibiting delayed embryonic development and/or from animals which generally yield low numbers of mature blastocysts after superovulation procedures or following natural mating.
  • BALB/c mice generally exhibit a low yield of embryos per mouse from naturally mated animals and a poor yield of blastocysts.
  • the present invention is, in its most preferred form, directed to mice strains exhibiting delayed embryonic development such as but not limited to BALB/c mice.
  • embryos are preferably isolated early on day 2, more preferably early on day 3 and still more preferably before approximately day 3V2, where day 0.5 is considered to be the time of conception after time 0 of mating. After this time, the yield of healthy embryos is greatly reduced. Accordingly, reference to an early embryo stage in BALB/c mice means embryos up to about day 2-3V2 post conception, and these general concepts apply to other mouse strains and other animals. Early embryos may also include compacted morulae. The embryos are cultured in vitro generally at 37°C in a CO 2 atmosphere. Generally, within 12 to 48 hours, and more preferably within about 24 hours, a substantial number of the cultured embryos develop into fully expanded blastocysts.
  • the embryos, blastocysts and ES cells may also be cultured in the presence of one or more active ingredients such as but not limited to one or more cytokines.
  • a particularly useful cytokine is leukemia inhibitory factor. See, for example, International Patent Application No. PCT/AU91/00307.
  • blastocysts from recipient animals having delayed embryonic development enables such animals to be used in chimeric animal production which increases the number of species or strains of animals that may be produced with well defined genetic backgrounds.
  • BALB/c embryos are, following the advent of the present invention, now easily accessible for the production of chimeras.
  • the methodology is employed to make mutations in particular genes in mice. These are referred to as gene knockout mice. Such mutant mice are particularly useful in studying the physiological effects of the mutations or for developing mice with a propensity to develop certain disease conditions such as cancer, diabetes, autoimmune disease or a range of other diseases.
  • another aspect of the present invention provides a genetically modified animal produced by isolating animal embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, animal ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant animal for development of genetically modified offspring.
  • a genetically modified animal produced by isolating animal embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, animal ES cells carrying a modified genomic sequence, implanting the blastocysts into a suitable implant animal for development of genetically modified offspring and then back-crossing a genetically modified off-spring with the same species, and preferably same strain, of animal from which the ES cells were isolated or an FI hybrid thereof and then selecting an off-spring exhibiting said modification.
  • a particular aspect of the present invention is directed to a genetically modified mouse produced by isolating mouse embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, mouse ES cells carrying a modified genomic sequence and then implanting the blastocysts into a suitable implant mouse for development of genetically modified offspring.
  • the present invention relates to a genetically modified mouse produced by isolating mouse embryos at an early embryonic stage, culturing said embryos in vitro until mature blastocysts and introducing into said blastocysts, mouse ES cells carrying a modified genomic sequence, then implanting the blastocysts into a suitable implant mouse for development of genetically modified offspring and then back-crossing a genetically modified off-spring with the same species of mouse, and preferably the same strain of mouse, from which the ES cells were isolated or an FI hybrid thereof and then selecting an off-spring exhibiting said genetic modification.
  • the ES cells genome are manipulated such that a particular gene or genes is/are targeted to remove or modify expression of the gene.
  • Recombinase systems are also useful in this respect.
  • the gene is a cytokine gene such as but not limited to tumour necrosis factor (TNF), an interferon (INF), a tyrosine kinase, leukaemic inhibitory factor (LIF), a colony simulating factor (CSF), lymphotoxin (LT), an interleukin (IL), a hormone and/or a neurological factor.
  • TNF tumour necrosis factor
  • INF interferon
  • LIF leukaemic inhibitory factor
  • CSF colony simulating factor
  • LT lymphotoxin
  • IL interleukin
  • Multiple, such as double or triple, mutations are also contemplated by the present invention such as but not limited to TNF/LT double mutations.
  • the recipient blastocysts for ES cell injection are from BALB/c mice or related to similar mice.
  • the present invention further contemplates gene-targeted mice produced using the method herein described.
  • the gene-targeted mice are mice such as but not limited to those incapable of producing TNF and/or LT- ⁇ or LT- ⁇ directly in the C57BL/6 mouse strain using ES cells derived from C57BL/6 mice.
  • the genetically modified mice (or other animals) of the present invention are particularly useful as in vivo animal models for assessing genetic effects on phenotype including the effects of gene function, expression and regulation on phenotype (7). They are also useful in vivo models, for example, in natural product and/or small molecule screening for agents which can alter, reverse or modify a particular engineered phenotype.
  • a genetically altered mouse with a propensity for developing cancer may be injected or contacted with a range of isolated products or synthesised chemical agents to screen for agents which prevent or delay onset of cancer.
  • a gene knock-out animal wherein said animal is produced by isolating animal embryos at an early embryonic stage, culturing said embryos in vitro until maturation to the blastocyst stage and introducing into said blastocysts, ES cells genetically modified by insertion, deletion and/or substitution of a genetic sequence and then implanting the blastocysts into a suitable implant animal for development of said gene knock-out animal.
  • the present invention is directed to a gene knock-out mouse wherein said mouse is produced by isolating mouse embryos at an early embryonic stage, culturing said embryos in vitro until maturation to the blastocyst stage and introducing into said blastocysts, ES cells genetically modified by insertion, deletion and/or substitution of a genetic sequence and then implanting the blastocysts into a suitable implant mouse for development of said gene knock-out mouse.
  • Still a further aspect of the present invention provides a genetically modified mouse produced by isolating embryos from BALB/c or related mice at an early embryonic stage, culturing said embryos in vitro until maturation to the blastocyst stage, introducing into said blastocysts, ES cells isolated from C57BL/6 or related mice wherein said ES cells are genetically modified by insertion, deletion and/or substitution of a genetic sequence and then implanting said blastocysts into an implant mouse such as but not limited to C57BL/6 or an FI hybrid thereof.
  • the present invention is related to a genetically modified mouse produced by isolating embryos from BALB/c or related mice at an early embryonic stage, culturing said embryos in vitro until maturation of said blastocysts, introducing into said blastocysts, ES cells isolated from C57BL/6 or related mice wherein said ES cells are genetically modified by insertion, deletion and/or substitution of a genetic sequence, implanting said blastocysts into an implant mouse such as but not limited to C57BL/6 or an FI hybrid thereof and then back- crossing a chimeric mouse produced by said method with a C57BL/6 mouse or a C57BL/6 x BALB/c FI hybrid mouse.
  • Another aspect of the present invention contemplates the method for isolating embryos from animals exhibiting delayed embryonic development or from animals which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, said embryos useful in the production of chimeric animals, said method comprising isolating said embryos from an animal at an early embryonic stage and then culturing said embryos in vitro until maturation into blastocysts.
  • the present invention contemplates the method for isolating embryos from mice exhibiting delayed embryonic development or from mice which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, said embryos useful in the production of chimeric mice, said method comprising isolating said embryos from a mouse at an early embryonic stage and then culturing said embryos in vitro until maturation into blastocysts.
  • Still a further aspect of the present invention contemplates a method of isolating embryos for use in the production of chimeric animals.
  • the animals are mice or other laboratory test animals such as but not limited to BALB/c mice or related mice.
  • a further embodiment is directed to the use of embryos isolated from animals exhibiting delayed embryonic development or from animals which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating, in the manufacture of blastocysts by isolating said embryos at an early embryonic stage from said animals and culturing said embryos in vitro until production of mature blastocysts for use in production of chimeric animals.
  • the animals are mice such as but not limited to BALB/c mice.
  • ES cells from a strain of mouse different to the BALB/c mouse are introduced into the BALB/c blastocysts and introduced into an implant mouse prior to production of chimeric off-spring.
  • the chimeric off-spring are back-crossed to the same species and more preferably same strain from which the ES cells are isolated or from a FI hybrid thereof.
  • compositions comprising animal embryos isolated at an early embryonic stage from animals exhibiting delayed embryonic development or from animals which generally yield low levels of mature blastocysts after superovulation procedures or following natural mating.
  • the composition would be in a form suitable for long term storage such as in a frozen form.
  • the animal embryos are mice embryos.
  • Embryos were harvested from BALB/c mice early (7am to 8am) on the third day (d3) after natural mating (dO). The timing is important as later on d3, the yield of healthy embryos was substantially reduced.
  • the uterus was removed and the two uterine horns were flushed as described (8, 9). Flushing of the uterus and embryo collection was performed with embryo- tested M2 medium (Sigma, St Louis, USA). Recovered embryos exhibited a stage of development varying between early and compacted morulae, with a few (-2%) of recovered cells being early blastocysts (Table 1).
  • Embryos from Example 1 were transferred to M16 medium at 37°C (Sigma, St Louis, USA) then incubated at 37 °C in a 5% v/v CO 2 atmosphere under embryo-tested mineral oil (Sigma, St Louis, USA). Within 24 hours, 70-80% of the cultured embryos were found to develop into fully expanded blastocysts. A further 10-20% developed into early blastocysts (Table 1) which were injected with ES cells last or reimplanted uninjected, together with injected blastocysts, to supplement the numbers of the latter and increase litter sizes (Table 2).
  • Transfected and G418-selected ES cells were thawed from stocks three days before injection and cultured basically as described (10) on neomycin-resistant foetal fibroblasts (11) in DMEM (high glucose and sodium pyruvate; Gibco BRL, UK) supplemented with 10% v/v FCS (pre -tested for ES cell culture as described (12)), glutamine (2mM; Gibco BRL, UK), NEAA's (O.lmM; Gibco BRL, UK), 0.1 mM ⁇ -mercaptoethanol (Sigma, St Louis, USA) and 200 U/ml leukaemia inhibitory factor (LIF) (ESGRO, Amrad, Australia).
  • DMEM high glucose and sodium pyruvate
  • Gibco BRL Gibco BRL
  • FCS pre v/v FCS
  • ES cells were passaged on the day before injection to disperse larger cell clumps and increase the growth rate of the culture.
  • ES cells were harvested and plated twice on gelatine-coated (0.1% w/v) petri dishes to remove fibroblasts and resuspended in DMEM-5% v/v FCS - 50 U/ml DNase (Sigma, St Louis, USA).
  • Twenty four hour cultured BALB/c blastocysts were micro-injected with ES cells using a Zeiss stereo-microscope at room temperature. Each blastocyst received between 8-12 ES cells.
  • blastocysts were reimplanted into pseudopregnant (d2.5) Foster mothers (implant animals). Eight blastocysts were implanted in one uterine horn and usually both uterine horns received blastocysts.
  • ES cells from C57BL/6 mice were subjected to genetic manipulation to inactivate the genetic loci for tumour necrosis factor (TNF) and/or lymphotoxin (LT)- ⁇ or LT- ⁇ (13).
  • TNF tumour necrosis factor
  • LT lymphotoxin
  • LT- ⁇ lymphotoxin- ⁇
  • LT- ⁇ LT- ⁇
  • MHC major histocompatibility complex

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne, en général, des animaux modifiés génétiquement et un procédé servant à obtenir ces derniers. Elle concerne, plus particulièrement, un procédé servant à obtenir des animaux modifiés génétiquement et consistant à introduire des cellules souches embryonnaires dans des blastocystes ayant été isolées à un stade embryonnaire précoce, puis cultivées jusqu'à un stade d'expansion totale in vitro. Ce procédé est particulièrement utile afin d'obtenir des blastocystes à partir soit d'animaux présentant un développement embryonnaire retardé, soit d'animaux ne produisant généralement que des nombres limités de blastocystes matures après des processus de superovulation ou à la suite d'accouplement naturel. Ce procédé permet également de générer une gamme d'animaux modifiés génétiquement. Ces animaux sont particulièrement utiles pour évaluer la fonction et l'expression d'un gène et leurs effets sur le phénotype de l'animal modifié.
PCT/AU1997/000817 1996-12-03 1997-12-01 Procede servant a obtenir des animaux modifies genetiquement Ceased WO1998024307A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51109/98A AU5110998A (en) 1996-12-03 1997-12-01 A method for producing genetically modified animals

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO4004A AUPO400496A0 (en) 1996-12-03 1996-12-03 A method for producing genetically modified animals
AUPO4004 1996-12-03

Publications (1)

Publication Number Publication Date
WO1998024307A1 true WO1998024307A1 (fr) 1998-06-11

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1997/000817 Ceased WO1998024307A1 (fr) 1996-12-03 1997-12-01 Procede servant a obtenir des animaux modifies genetiquement

Country Status (2)

Country Link
AU (1) AUPO400496A0 (fr)
WO (1) WO1998024307A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1176189A1 (fr) * 2000-07-21 2002-01-30 Fornix Biosciences N.V. Cellules de type cellules souches

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119184024A (zh) * 2024-08-27 2024-12-27 南京农业大学 一种提高动物早期胚胎定植数量和促进胚胎发育的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LABORATORY ANIMAL SCIENCE, 43(1), (1993), KOVACS M.S. et al., "Use of Superovulated Mice as Embryo Donors for ES Cell Injection Chimeras", pages 91-3. *
METHODS IN ENZYMOLOGY, 225, (1993), STEWART C.L., "Production of Chimeras Between Embryonic Stem Cells and Embryos", pages 823-55. *
METHODS MOL. BIOL. (TRANSGENESIS TECHNIQUES), 18, (1993), WELLS D., "Production of Chimeras Derived from Murine Embryonic Stem Cells", pages 217-37. *
NUC. ACIDS. RES., 25(4), (1997), LEMCKERT F.A., "Gene Targeting in C57BLI6 ES Cells. Successful Germ Line Transmission Using Recipient BALB/c Blastocysts Developmentally Matured In Vitro", pages 917-8. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1176189A1 (fr) * 2000-07-21 2002-01-30 Fornix Biosciences N.V. Cellules de type cellules souches
WO2002008388A3 (fr) * 2000-07-21 2002-05-02 Fornix Biosciences N V Cellules du type cellule souche

Also Published As

Publication number Publication date
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