EP1009816A1 - Herstellung von transgenischen donorzellen zum kerntransfer - Google Patents

Herstellung von transgenischen donorzellen zum kerntransfer

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Publication number
EP1009816A1
EP1009816A1 EP98907009A EP98907009A EP1009816A1 EP 1009816 A1 EP1009816 A1 EP 1009816A1 EP 98907009 A EP98907009 A EP 98907009A EP 98907009 A EP98907009 A EP 98907009A EP 1009816 A1 EP1009816 A1 EP 1009816A1
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EP
European Patent Office
Prior art keywords
cell
cells
animal
transgene
embryo
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EP98907009A
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English (en)
French (fr)
Inventor
Alexander Jarvis Kind
Angelika Elisabeth Schnieke
Ian Garner
Alan Colman
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PPL Therapeutics Scotland Ltd
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PPL Therapeutics Scotland Ltd
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Publication of EP1009816A1 publication Critical patent/EP1009816A1/de
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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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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)
    • 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
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • the present invention relates to a process for producing a nuclear donor cell, a process for obtaining predictive information of the phenotype of a transgenic animal and a process for reconstituting an animal embryo, wherein all processes involve the screening of a cell and/or analysis of the phenotype of a cell, preferably analysis of transgene expression characteristic (s) .
  • the present invention also provides nuclear donor cells, reconstituted animal embryos and animals according to the process of the invention.
  • Transgenic animals have been defined as "animals that have integrated foreign DNA into their germ-line as a consequence of experimental introduction of DNA” (Palmiter RD, and Brinster R . Cell , 41, 343-345, 1985) .
  • transgenic in a broader sense to include animals in which any type of genetic modification (eg. gene deletion, mutation, substitution) has been carried out using, at some stage, genetic manipulation in vi tro .
  • a description of the present invention is preceded by a review of currently available and prospective methods of producing transgenic animals.
  • Pronuclear microinjection This method was first described in 1980 (Gordon J et al . , Proc . Natl . Acad . Sci . USA 77: 7380-7384), and since then has been used extensively in many species. In short, naked DNA is microinjected into the pronucleus of explanted zygotes which are then transferred to foster mothers to complete gestation. Transgene integration into the host genome is random and in mice typically occurs in 5-20% of offspring.
  • Pronuclear microinjection is by far the most widely used method of gene transfer in livestock (Ebert KM and
  • mice the transgenic rate ( ⁇ l-5% of offspring) is often significantly lower than mice and the costs considerably greater. It has been estimated that approximately 1200 microinjected bovine zygotes are required to produce a single transgenic calf (Eyestone, .H., Reprod . Fertil . Dev. , 6, 647-652, 1994). A straightforward application of the approach used in mice therefore requires approximately 300 super-ovulated cows as zygote donors and 600 cows as recipients for microinjected embryos. Although methods have been developed to reduce these numbers, there is still a considerable incentive to use animals more efficiently.
  • transgenic embryos can be minimised by identifying transgenic embryos either before transfer or during gestation.
  • Polymerase Chain Reaction (PCR) analysis of embryo biopsies obtained prior to embryo transfer has been used to produce transgenic cattle (Haskell and Bowen, Mol . Reprod . Dev. 40 , 1994).
  • PCR Polymerase Chain Reaction
  • there is no method available which can reliably distinguish between integrated transgene and residual non-integrated DNA Krisher, R.K., et al . , Theriogenology 41, 229, 1994
  • transgenic fetuses can be identified in utero from samples obtained by amnio - or allantocentesis .
  • Retrovirus mediated gene transfer Infectious retroviral vectors derived from replication defective retroviruses can be used to transduce non-viral genes with high efficiency, into dividing cells in vivo or in vi tro (Weiss, R. , et al . , RNA tumor viruses . Cold
  • Retrovirus mediated gene transfer has been used to produce transgenic animals of several species, e.g. mice
  • Retroyiruses do, however, suffer several disadvantages which severely limit their usefulness.
  • the size of DNA transduced is limited and effectively restricts the use of retroviral vectors to cDNAs, which are generally poorly expressed as transgenes .
  • Differential timing of retroviral integration and the possibility of several different independent integrations leads to the frequent production of mosaic animals, which can fail to transmit the transgene through the germ line.
  • Insertion of retroviral long terminal repeats (LTRs) into the host genome can also cause activation of adjacent genes with possible deleterious effects.
  • LTRs long terminal repeats
  • Perhaps the most serious problem with transgenic animals carrying retroviral vectors is the risk of producing replication competent virus by recombination. Uncertainty regarding this possibility excludes at present, the use of animals containing retroviral transgenes for most human applications .
  • DNA internalisation into the sperm head has also been shown in mouse and cattle (Bachiller et al . , op. ci t . 1991; Atkinson, op . ci t . 1991) .
  • Embryonic stem cell mediated transgenesis Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo (Evans, M.J., and Kaufmann, M.H. , Nature 292, 154-156, 1981; Martin, G.R., Proc . Na tl . Acad . Sci . USA 78, 7634-7638, 1981) .
  • ES cells can be grown and manipulated in vi tro and then introduced into a pre-implantation stage host embryo by microinjection or aggregation, where they participate in the formation of a chimeric animal and can contribute to somatic and germ cell lineages.
  • ES cells provided the first cell mediated method of transgenesis .
  • ES cells are a direct alternative to microinjection into zygotes for the production of random transgenics and have been used where DNA microinjection is problematic, eg. in the production of mice containing yeast artificial chromosomes (Pearson, B.E., and Choi, T.K., Proc . Na tl . Acad . Sci . USA 90, 10758-62., 1993; Jakobovits, A., et al . , Nature 362, 255-258, 1993).
  • ES cell mediated transgenesis exploits the ability of ES cells to support homologous recombination between exogenous DNA and chromosomal sequences at a relatively high frequency.
  • Gene targeting by homologous recombination allows precise modifications to be made at predetermined sites in the genome and has been used extensively to effect a wide variety of genetic manipulations (reviewed by Ramirez-Solis, R. and Bradley, A., Curr. Opin . Biol . 5, 528-533, 1994 and by Brandon, E.P., et al . , Curr. Biol . 5, 5, 625-634, 758-765, 873-881, 1995).
  • Theriogenology 41, 321, 1994 cattle (Sims, M.M. and First, N.L., 1993 Theriogenology 39, 313, 1993; Cherny, L.R., and Maus, J . , Theriogenology 41, 175, 1994; Stice, S., et al . , Theriogenology 41, 304, 1994; Strelchenko, N. and Stice, S., Theriogenology 41, 301, 1994), pig (Piedrahita, J.A., et al . , op . ci t . 1990; Notorianni, E., et al . , J. Reprod . Fertil .
  • Embryonic germ cells are the progenitors of the gametes. Matsui et al . , ( Cell 70, 841-847, 1992) and Resnick et al . , (Nature 359, 550- 551, 1992) first identified a combination of growth factors (Leukaemia inhibitory factor, steel factor and basic fibroblast growth factor) which promote long term growth of mouse PGCs and their conversion to a cell type termed embryonic germ (EG) cells. EG cells closely resemble ES cells and are functionally equivalent for cell mediated transgenesis. EG cells can be manipulated in vi tro, and then contribute to somatic and germ cell lineages of chimeric animals (Stewart, l.C, Dev. Bio . 161, 626-628, 1994; Labosky, P., et al . , In : GexV iine development : pp 157-178, Pub. Wiley, Chichester, 1994) .
  • mice There has been a recent demonstration that transplanted spermatogonia can repopulate the testes of sterile, or sub-fertile recipients in mice (Brinster, R.L. and Zimmermann, J.W., Proc . Natl . Acad . Sci . USA 91, 11298-11302, 1994; Brinster, R.L. and Avarbock, M.R., Proc . Natl . Acad . Sci . USA 91, 11303-11307 and rat (Avarbock, M.R. Nat . Med .
  • liver transfer in mammals has only been possible using nuclei from cells obtained directly from early embryos, or subjected to very short periods in culture.
  • Live animals have been produced by nuclear transfer from embryonic blastomeres into enucleated oocytes in pigs (Prather, R.S., et al . , Biol . Reprod . 41, 414-418, 1989), cattle (Prather, R.S., et al . , Biol . Reprod . 41: 414-418, 1987; Bondioli, K.R. et al . , Theriogenology 33, 165-174., 1990; Keefer, C.L. et al . , Biol . Reprod .
  • Mid-gestation bovine fetuses have been produced by nuclear transfer from cultured bovine embryo explants (Stice, S., et al . , op . ci t . , 1994; Strelchenko, ⁇ . and Stice, op . ci t . 1994).
  • mice Difficulties associated with mice may be related to the time at which the embryonic genome becomes transcriptionally active.
  • Mouse embryos initiate transcription at the 2 cell stage, pigs at 4 cell, cows at 4-8 cell and sheep at the 8-16 cell stage (Prather, R.S., J. Reprod . Fertil . 48, 17-29, 1993). It is conceivable that the delay before genome activation allows the transplanted nucleus to be reprogrammed by its cytoplasmic environment.
  • transgenic animals random integration of foreign DNA into the host chromosome is the norm. Integrated transgenes typically occur as single copies, or tandem head to tail arrays at a single site apparently randomly located within the genome. Transgenes integrated at different sites can vary widely in expression level due to the influence of the local genetic environment (Wilson. C et al . , Ann. Rev. Cell Biol. 6, 679-714, 1990). This position effect variation requires that many lines of transgenic large animals must be independently derived to obtain one with a suitable level of expression.
  • Gene targeting frequency has been shown to be increased by introducing a double strand break at the target locus by a site-specific endonuclease in human somatic cells and murine ES cells (Brenneman, M. , et al., Proc . Natl . Acad. Sci . USA, 93, 3608-3612, 1996) (Smih, F. et al . , 1995, Nuc . Acid Res. 23, 5012-5019).
  • endonucleases e.g.
  • I-Scel which cleave at rare (18bp) recognition sites (Jacquier, A., and Dujon, B., Cell , 41, 383-394, 1985) allow double-strand breaks to be created at unique target loci in the host genome (Rouet, P., et al . , Mol . Cell . Biol . 14, 8096-8106, 1994).
  • Donor cells can be genetically manipulated and optionally screened in vi tro for the required genotype (ie. stable integrant) .
  • the chosen genotype can be transferred to whole animals by nuclear transfer. This approach is particularly suitable for large animals because, in contrast with ES cells, the animals are derived completely from the donor cell, not chimeras.
  • a problem associated with selecting donor cells on the basis of genotype is that genotype does not necessarily give information about the phenotype of the cell or of an animal derived from that cell.
  • selection of donor cells on the basis of genotype is not an effective route for the production of transgenic animals with a desired phenotypic trait. It has not previously been thought possible to analyse the phenotype of nuclei which are to be used in the production of transgenic animals.
  • the present invention describes how it is possible to obtain such phenotypic information of nuclei to be used in the production of transgenic animal and thus describes a significant improvement in the production of transgenic animals with a desired phenotypic trait. Furthermore, it is shown that nuclear transfer from cultured cells is not limited to a particular cell type such as that described by Campbell et al . , (op . ci t . , 1996) . As is demonstrated, cells of various tissues can be used successfully. This provides the major advantage that a variety of donor cells can be chosen to provide predictive information about transgene expression in the whole animal .
  • transgene construct is introduced into cultured cells, cell clones are derived and individually analysed for transgene expression. Clones selected for desirable transgene expression are then used as nuclear donors to produce transgenic animals .
  • the present invention provides, as a first aspect, a process for producing a nuclear donor cell, the process comprising, transfecting a cell with a transgene and screening the cell for a desired phenotype.
  • transgene includes any type of genetic modification (deletion, mutation, substitution) and includes integration of foreign DNA and of endogenous DNA.
  • transfection and transforming mean the introduction of nucleic acid (usually DNA) into a cell by any means, including micro injection and other techniques described in the introductory section.
  • the process for producing the nuclear donor cell may further include assessing the cell for suitability for nuclear transfer, such as testing the cell for its ability to survive serum starvation and/or establishing a normal chromosomal complement.
  • the donor cell may be, but does not have to be, in culture.
  • the screening of the cell for a desired phenotype is preferably carried out in vi tro but may also be carried out in vivo .
  • the screening for the desired phenotype is preferably to determine, or to screen for transgene expression characteristics. This is a specific part or area of a cell's phenotype. It most accurately reflects information (qualitative and quantitative) about the transgene.
  • the determination or screening of/for transgene expression characteristics is preferably by analysis of transgene RNA expression or by analysis of transgene protein expression. Such analysis can give qualitative and quantitative information concerning the transgene.
  • the cell for transfection and screening may be any cell, including adult somatic cells, embryonic somatic cells or foetal cells, which can act as competent nuclear donor cells.
  • the invention relates to at least a partially differentiated cell (e.g. post-embryonic stage) and fully differentiated cells.
  • the transfected cell naturally models a tissue of interest or is genetically engineered to model a tissue of interest.
  • the cells may require addition of a suitable stimulus in order to model a tissue of interest.
  • the ability to respond to a particular stimulus may be required.
  • This can include a portion of the transgene construct.
  • the present invention includes the modification of cells so that they can provide a predictive model. For example, any modification can be made where genes are added to confer particular tissue specific properties. Specific embodiments include the expression using the prolactin signally pathway
  • genetic modification may involve the introduction of those components of a signal transduction pathway required for tissue specific gene expression.
  • Signal transduction pathways for tissue specific gene expression in several tissues have been elucidated in detail, e.g. prolactin induced gene expression in mammary epithelium, interleukin 2 induced gene expression in lymphocytes and iron induced gene expression in a wide variety of cell types.
  • genetic modification may be achieved by the introduction of key regulatory factors which induce tissue specific gene expression. Examples of this are the muscle transcriptional activators myoD
  • the process for producing the nuclear donor cell may further include the step of preparing the cell for nuclear transfer, e.g. by inducing the nuclear donor cells into a quiescent state, or synchronising the cell component activities, or any other method.
  • the process may also include a step of transgene placement at a favourable site.
  • This process is summerized here with a full description given under the advantages of the invention following a discussion of the particular aspects.
  • the transgene for transfecting the cell may be flanked by a recognition site for a site specific recombinase and a recognition site for a rare cutting endonuclease .
  • An example of the site specific recombinase is the bacteriophage PI Cre recombinase LoxP site and an example of a rare cutting endonuclease site is the site for I-Sce I.
  • Such a transgene construct enables replacement of the first transgene with a second transgene at a specific target locus .
  • a nuclear donor cell produced by a process of the first aspect of the invention.
  • Such a nuclear donor cell can be used for nuclear transfer, for example as previously described by Campbell, K.H.S. et al . , op . ci t . 1996 and in WO97/07669 the content of which is fully incorporated herein by reference. All preferred features of the first aspect also apply to the second aspect .
  • a process for obtaining predictive information of a phenotype of a transgenic animal comprising transfecting a cell with a transgene and screening the cell to obtain phenotypic information.
  • the process according to the third aspect of the invention may further include preferred features according to the first or second aspect of the invention.
  • a process for reconstituting an animal embryo comprising, transfecting a cell with a transgene, analysing the phenotype of the cell, inducing the cell into a state suitable for nuclear transfer, such as a quiescent state and transferring the nucleus of the cell into a suitable recipient cell .
  • a basic process for reconstituting an animal embryo is described by Campbell, K.H.S. et al . , op. cit. 1996 and in WO97/07669.
  • the recipient host cell is preferably an enucleated metaphase II oocyte, an enucleated unactivated oocyte or an enucleated preactivated oocyte. Enucleation may be achieved physically, by actual removal of the nucleus, pro-nuclei or metaphase plate, or functionally, such as by the application of ultraviolet radiation or another enucleating influence.
  • Suitable recipient enucleated oocytes are : Metaphase Arrested Gl/GO Accepting Cytoplast; GO/Gl Activation and
  • the nucleus of the donor requires transfer to the recipient cell.
  • This can be established by fusion (e.g. exposure of cells to fusion-promoting chemicals, such as polyethylene glycol , the use of inactivated virus (such as Sendai virus) or by the use of electrical stimulation) or by other techniques such as microinjection (Ritchie and Campbell, J. Reproduction and Fertility, Abstract Series No. 15, p60) .
  • Preferred analysis of the phenotype of the cell is by analysis of transgene RNA expression or analysis of transgene protein expression. Such analysis can give qualitative and quantitative information concerning the transgene. All preferred features of the first to third aspects also apply to the fourth.
  • the cell is a mammary epithelial cell (foetal or other) , a fibroblast cell, an endothelial cell or a sub-endothelial cell.
  • all aspects of the invention are applicable to all animal cells and animals including birds, such as domestic fowl, amphibian species and fish species.
  • the present invention is most applicable to non-human animals, especially non- human mammals, and placental mammals.
  • ungulates such as cattle, sheep, goats, water buffalo camels and pigs that the invention is likely to be most useful.
  • the invention is also likely to be applicable to other economically important animal species such as, for example, horses, llamas or rodents e.g. rats, mice or rabbits.
  • the process according to the fourth aspect of the invention reconstitutes an ungulate species embryo, more preferably a non-human, such as a cow, bull, pig, goat, sheep, camel or water buffalo animal embryo .
  • a non-human such as a cow, bull, pig, goat, sheep, camel or water buffalo animal embryo .
  • a suitable recipient cell for the fourth aspect of the invention is an oocyte.
  • the oocyte is advantageously enucleate .
  • the transfected cell models a tissue of interest, either naturally, or by genetic modification, as described above according to the first aspect of the invention.
  • the transgene of the cell can be used to place an alternative transgene at a favourable site.
  • the transgene may be flanked by a recognition site for a site specific recombinase and a recognition site for a rare cutting endonuclease, as described according to the first aspect of the invention.
  • the fifth aspect of the invention provides a reconstituted animal embryo produced by a process according to the fourth aspect of the invention.
  • the reconstituted animal embryo may be developed to term to produce a transgenic animal.
  • Such an animal is a sixth aspect of the present invention. All preferred features of aspects one to four also apply to aspects five and six.
  • the seventh aspect of the invention provides a process for producing an animal, the process comprising, reconstituting an animal embryo according to the process of the fourth aspect of the invention, causing such an animal to develop to term from the embryo and optionally breeding from the animal. All progeny/offspring from any animal of the present invention is also covered and is part of the present invention. Accordingly any animal derived from any animal of the invention is also part of the invention.
  • the step of causing the animal to develop to term may be done directly or indirectly.
  • direct development the embryo is allowed to develop to term without further intervention beyond that necessary to allow development to term to take place.
  • Indirect development includes further manipulation of the embryo before full development takes places.
  • One example of such a manipulation includes splitting the embryo and the cells clonally expanded for the purposes of improving yield.
  • An eighth aspect of invention provides an animal produced by the process of the seventh aspect of the invention.
  • a ninth aspect of the invention provides a process for targeting a transgene to a location of interest .
  • the process may comprise: selecting a nuclear donor cell produced by the second aspect of the invention, specifically, wherein the transgene is flanked by a recognition site for a site specific recombinase and a recognition site for a rare cutting endonuclease; or selecting a reconstituted animal embryo which has been produced by the fourth aspect of the invention which includes a transgene which is flanked by the same loci; or an animal developed from such a reconstituted animal embryo, and using a gene targeting vector, preferably isogenic to the host cell, to place a target transgene at the target locus.
  • Other processes for targeting a transgene to a location of interest may be used.
  • a tenth aspect of the invention provides a nuclear donor cell, a reconstituted embryo or an animal produced by the ninth aspect of the invention.
  • Predictive information on transgene expression can be gained from cells of the species of interest, rather than from another species, typically mouse.
  • Transgenic animals produced from a cell clone are genetically identical, thus phenotypic variation can be minimalized.
  • keratinocytes Watt, F.M. FASEB J. , 5, 287-294, 1991, Dubertret, L. Skin Pharmacol . 3, 144-148, 1990
  • hepatocytes Ulrich, R.G. et al . , Toxicol . Letters, 82-83, 107-115, 1995
  • myoblasts Daubas, P., et al . , Nuc . acids res . 16, 1251-1271, 1988. Therefore a wide variety of cell types from many species are available as potential predictors of transgene expression and nuclear donors .
  • a preferred embodiment of the invention is the use of cultured cells to predict lactation specific expression in the mammary gland of transgenic animals.
  • vi tro models of mammary epithelium have been established in mouse (Barcellos- Hoff, M.H., et al . , Development 105, 223-235 1989; Seely, K.A. and Aggeler, J., J. Cell . Physiol . 146, 117- 130, 1991), sheep (Finch, L.M.B. et al . , Biochemical Society Transactions 24, 3695, 1996) and goat (Wilde, C.J. et al . , Biochemical Journal 305, 51-58).
  • transgenic animals can be made by conventional pro-nuclear injection and nuclear-transfer cell lines can be derived from animals shown to express a transgene optimally in the tissue of interest (e.g. mammary gland) . If the location of the transgene (by conventional techniques or techniques according to the present invention) is judged to be favourable for expression, there are several possible schemes by which other transgenes can be placed at the same site.
  • a site which supports favourable expression is identified using a transgene construct linked to a counter-selectable marker, e.g. the Herpes simplex tk gene, loss of which can be selected by the drug ganciclovir or the HPRT (Hypoxanthine phosphoribosyl transferase) gene, loss of which can be selected by the drug 6-thioguanine in cells lacking endogenous HPRT activity, or the Aequoria Victoria green fluorescent protein gene (Chalfie, M. et al . , 1994, Science 263, 802-805) loss of which can be detected visually.
  • DNA regions flanking the integrated transgene are cloned and incorporated into a replacement gene targeting vector.
  • Somatic cells containing a transgene plus- marker construct at the desired site are derived either from the original clone, or re-derived from a transgenic animal produced from that clone. Re-derivation of early passage cultures may be preferable as this minimises the time in culture and any consequent deleterious effect on nuclear transfer.
  • a site which supports favourable expression is identified using a transgene construct which is flanked by recognition sites for a site specific recombinase (e.g. bacteriophage PI Lox P sites recognised by Cre recombinase Sauer, B., Methods Enzymol . 225, 890-900, 1993) , or yeast FRT sites recognised by FLP recombinase (Flering et al . , Proc . Natl . Acad . Sci . USA . , 90, 8469- 8473, 1993)) . These are flanked on one side by a recognition site for a rare cutting endonuclease, e.g. I-Scel.
  • a site specific recombinase e.g. bacteriophage PI Lox P sites recognised by Cre recombinase Sauer, B., Methods Enzymol . 225, 890-900, 1993
  • LoxP sites and I-Scel site have been used.
  • a counter-selectable marker could be included within the region flanked by the recombinase recognition sites, but, given the high efficiency of recombinase mediated excision, may not be necessary.
  • Somatic cells containing a transgene construct at the desired site are derived either from the original clone, or re-derived from a transgenic animal produced from that clone .
  • the first transgene is excised by the action of Cre recombinase, which can either be introduced into cells as protein, or expressed from a transfected DNA construct .
  • a gene targeting vector (possibly including a selectable marker e.g. neo) is used to place a second transgene at the target locus.
  • I-Scel endonuclease introduced into cells with the targeting vector, is used to enhance the efficiency of targeting by double strand cleavage at the I-Sce site at the target locus.
  • Illustrated in Figure 1 is a replacement type vector, alternatively an insertion type vector could be used.
  • Cells confirmed as containing a transgene at the target locus are used to generate a second transgenic animal .
  • the present invention contemplates the production of transgenic animals which carry antibiotic resistance. Such transgenic animals may be undesirable because of the risk that drug resistance may be transferred to pathogenic prokaryotic organisms. Several possibilities exist to reduce this risk.
  • the selectable marker is removed from cells in vi tro before nuclear transfer. This can be achieved by excision at flanking site specific recombinase recognition sites, e.g. the bacteriophage PI Cre LoxP system (Sauer, B., Methods . Enzymol . 225, 890-900, 1993). Removal of a selectable marker by this method would, of course, use a site specific recombinase system different from any involved in transgene replacement .
  • DNA transfer is carried out by a method which does not require drug selection, e.g. DNA microinjected into cells, individual clones derived and assayed for the presence of the transgene.
  • the selectable marker used can be modified such that it is nonfunctional in prokaryotic cells.
  • the neo expression cassette used in the PGK neo construct already lacks 5 ' elements required for bacterial expression. This gene could be modified further by the introduction of a eukaryotic intron to render it non-functional in prokaryotes .
  • Selectable markers can be used which are nonfunctional in prokaryotes, e.g. histidinol selection for the his gene (Hartman, S., et al . , Proc . Natl . Acad . Sci USA, 77, 3567-3570, 1980) .
  • the single recombinase recognition site left after step 3 in the scheme shown in Figure 1 can be used to target integration of a second transgene to that site by site-specific recombination.
  • a second transgene construct containing a single recombinase recognition site is introduced into the cells as a circular episome. Recombinase mediated recombination between these two sites results in integration.
  • the efficiency of this reaction relative to the reverse excision reaction has not been ' clearly established, but integration of a transgene into cultured cells by this method has been reported (Rucker, E.B. et al . , Theriogenology 47 , 228, 1997).
  • Each of the 3 panels shows polymerase chain reaction amplification products from genomic DNA samples. The identity of each microsatellite sequence is indicated to the right.
  • PCR products were separated by electrophoresis on 4% metaphor agarose (FMC) and visualised by ethidium bromide fluorescence.
  • FMC metaphor agarose
  • PCR primers and reaction conditions used were as described by Buchanan, F.C. et al . , (Mammalian genome 4, 258-264; 1993).
  • Figure 3
  • Each lane shows amplification products from samples of genomic DNA using a combination of two primer pairs: one designed to amplify a portion of the SRY gene present on the Y chromosome, and one designed to amplify a portion of the ZFX gene present on the X chromosome.
  • the presence of two amplification products indicates a male
  • one amplification product indicates a female.
  • Lanes A, B male and female control DNA; lanes C-I, embryo transfer recipients; lane J, SEC1 cells; lane K-N lambs born from SEC1 nuclear transfer; lane O, BLWF1 cells; lanes P, Q, lambs born from BLWF1 nuclear transfer. Individual identification numbers of each sheep are shown over each lane .
  • PCR products were separated by electrophoresis on 2% agarose and visualised by ethidium bromide fluorescence. Primer sequences and amplification conditions were as described by Griffiths, R. et al . , (Molecular Ecology, 2, 405-406, 1993) and Horvat, S. et al . , (Transgenic Research, 2, 134-140, 1993) .
  • Example 1 Live sheep produced by nuclear transfer from fetal fibroblast cells
  • Fibroblast cells termed BLWF1
  • BLWF1 Fibroblast cells
  • BLWF1 cells at passage 4-6 were prepared for nuclear transfer by serum staravation and nuclear transfer carried out as described by Campbell et al . , (op. cit., 1996) results are shown in Table 1:
  • This example demonstrates the production of transgenic sheep by cell mediated transgenesis using fetal fibroblasts.
  • PDFF Poly Dorset Fetal Fibroblast 1-7
  • Stocks of early passage PDFF cells were cryopreserved to provide cells when required.
  • Ovine fetal fibroblast cells are particularly suitable for cell mediated transgenesis and nuclear transfer because they are readily available in large numbers, grow rapidly and sustainably in culture for 12 weeks, are readily transfectable, maintain euploidy after long term culture (Table 2) and survive serum starvation.
  • PDFF2 cells were stably transfected with a milk specific transgene pMIXl and a selectable marker, clones were derived, analysed and suitable clones used as nuclear donors to produce transgenic shee .
  • the procedures used were as follows:
  • pMIXl consists of the human Factor IX gene (31.37Kb), from 12bp upstream of the translational start signal to 12bp downstream of the translational stop signal, flanked at the 5 1 end by a 4.2Kb BLG promoter region and at the 3' end by a 2.2Kb BLG region containing 3 ' untranslated sequence polyadenylation signal and non-transcribed region. This is contained within the bacterial pUC18 vector.
  • pMIX was cotransfected with a selectable marker construct PGKneo in which the neo gene, conferring resistance to the drug G418 (Colbere-Garapin et al . , J. Mol . Biol . 150, 1-14, 1981) is under the control of the human phosphoglycerate kinase promoter.
  • cells were treated in two ways. One group was grown at high density under G418 selection, then cryopreserved as a pool for nuclear transfer. The other group was plated at low density under G418 selection and cloned transfectants grown from isolated colonies. 48hrs after transfection cells were split 1:10 and G418 added to 0.6mg/ml . Transfected PDFF2 cells reached subconfluence after 6 days selection. At third passage one portion of the cells was split 1:10, subjected to a further 5 days selection and cryopreserved as an uncloned population.
  • Liveborn lambs were defined as those with a heart beat and able to breath unassisted at birth.
  • This example demonstrates how cultured cells can be modified to provide predictive information on transgene expression and how that information can inform the choice of transfected cell clones used for nuclear transfer.
  • fetal fibroblast cells are suitable for cell mediated transgenesis, they are unlikely, without modification, to express genes designed for expression in mammary epithelium. However, such cells can be genetically modified to support milk gene expression. This has been previously demonstrated with other cell types which do not normally express milk genes.
  • Chinese hamster ovary cells can be stably transfected with the rabbit prolactin receptor and support expression of the mammary specific rat ⁇ -casein and ovine ⁇ -lactoglobulin promoters (Lesueur, L. et al . , Proc . Natl . Acad . Sci . USA 88,824-828, 1991; Demmer, J. et al., Mol . Cell .
  • NIH3T3 mouse fibroblasts can be transfected with the human prolactin receptor and support expression of the rat ⁇ -casein promoter (Das, R. and Vonderhaar, B.K., Mol . Endocrinol . 9, 1750-1759, 1995).
  • fetal fibroblast cells can be modified to support the expression of milk genes.
  • Table 5 shows mammary specific reporter gene expression from fetal fibroblasts modified by the addition of cloned components of the prolactin signal transduction pathway in two repeat experiments.
  • PDFF2 cells were stably cotransfected with constructs designed to express the prolactin receptor (long form of the rabbit prolactin receptor cDNA driven by SV40 promoter early regions) and mouse STAT5a (STAT5a cDNA driven by a CMV promoter) .
  • a construct composed of the firefly luciferase gene under the control of the ovine ⁇ - lactoglobulin promoter was used to provide a rapid indication of milk protein promoter driven gene expression.
  • fibroblasts with mammary epithelial cells.
  • Milk ' protein gene expression can be induced in stably transfected fibroblasts by fusion with mammary epithelial cells which are functionally differentiated or capable of undergoing functional differentiation. In this way cytoplasmic and cell surface components required for the activation of milk protein gene expression are made available to the fibroblast nuclei.
  • the heterokaryon fusion products can be screened directly for expression of the transgene.
  • PDFF2 cells stably transfected with a construct comprising the green fluorescent protein (GFP) (Chalfie et al . op. cit., 1994) gene under the control of the ⁇ - lactoglobulin promoter were fused with a conditionally transformed murine epithelial cell line KIM-2, which has previously been demonstrated as capable of expressing a broad range of milk proteins in vi tro
  • transfected PDFF2 cells and KIM-2 cells were fused by co-centrifugation in the presence of a fusogenic reagent e.g. polyethylene glycol 1000, 6000, or inactivated Sendai virus.
  • a fusogenic reagent e.g. polyethylene glycol 1000, 6000, or inactivated Sendai virus.
  • Conditions for heterokaryon formation were optimised by assaying the number of heterokaryons formed.
  • Murine and ovine nuclei in heterokaryotic cells were identified by staining with Hoechst 33258, which produces characteristic different patterns of nuclear staining in these species. Following induction with lactogenic hormones the expression of the transfected gene expression from fibroblast nuclei was visualised as GFP fluorescence in heterokaryons
  • Ovine mammary epithelial cells were prepared from mammary tissue by a method originally developed in mouse by Barcellos-Hoff and Bissell (Development 105, 223-235, 1989).
  • the excised glands from pregnant or lactating animals were minced into l-2mm fragments and incubated with digestive enzymes such as collagenase and dispase to dissociate the tissue. This released loosely associated fibroblasts and adipocytes . Differential centrifugation was used to enrich for the epithelial component.
  • the cellular composition of the cultures was established on the following criteria: cell morphology, immunohistochemical detection of epithelial cell-specific cytokeratin markers such as keratin 18 and 19 (Lane, E.B. J.
  • Ovine mammary epithelial cells were fused with PDFF fetal fibroblasts stably transfected with a ⁇ - lactoglobulin driven GFP reporter construct.
  • Application of lactogenic stimuli resulted in GFP expression in heterokaryons, indicating induction of milk gene expression.
  • PDFF cell clones transfected with a transgene construct were isolated, a sample of each was cryopreserved at an early stage, and a portion grown on for analysis for suitability for nuclear transfer as described in example 2. A further round of screening was then carried out to determine the level of transgene expression in vi tro . Samples of each fibroblast clone were modified to allow induction of BLG directed transgene expression. The level of transgene expression in each clone was assayed by RNA and protein analysis and those clones which displayed the highest levels of expression were identified. The unmodified frozen sample corresponding to those clones was then thawed and cells used as a nuclear donors as described in example 2.
  • Example 4 Placement of transgenes at optimal sites This example demonstrates how transgenes can be placed at genomic locations previously identified as supporting favourable transgene expression.
  • Cell mediated transgenesis is an ideal way to identify locations in the genome which are favourable to transgene expression. Far more individual integration sites can be screened in transfected cells than is feasible in transgenic animals. Once a location shown to support high expression has been identified, other transgenes can be targeted to the same position.
  • a transgene construct was generated which was flanked by recognition sites for a site specific recombinase (e.g. bacteriophage PI Cre recombinase LoxP sites) and a recognition site for a rare cutting endonuclease (e.g. I -See I) .
  • the construct was transfected into cells, independent transfected clones were screened for transgene expression as described in example 3 and a favourable clone used to generate transgenic animals by nuclear transfer as described in example 3. This provided confirmation of the ability of the cell clone to produce a healthy animal by nuclear transfer.
  • transgenes can then be targeted to the same site in cells in vi tro using the scheme outlined in Figure 1.
  • Example 5 Sheep produced by nuclear transfer from embryo derived cells
  • This example demonstrates the production of sheep by nuclear transfer from another cell type, providing further evidence of the general applicability of the present invention.
  • a Poll Dorset sheep embryo was flushed from the oviduct of a ewe at 9 days post coitum and placed in culture.
  • the embryonic disc was isolated by microdissection and cultured in the presence of mitotically inactivated feeder cells. After 8 days the explanted embryonic disc was disaggregated and cells replated onto a fresh feeder layer After a further 7 days growth a colony of extended very flat cells became apparent, this was isolated and grown further in the absence of feeder cells.
  • the cells derived are termed SECl and do not resemble the TNT4 cells derived by a similar procedure by Campbell et al . , ( op.
  • SECl cells were prepared for nuclear transfer by serum starvation for 5 days and nuclei transfer carried out as in example 2.
  • Table 7 shows the results of the nuclear transfer experiment .
  • This example demonstrates how transgene analysis can be facilitated in short lived cells by conditional immortalisation of a portion of each transfected clone.
  • SECl cell mediated transgenesis is hindered by the relatively short lifespan of the cells in culture. SECl cells grow for approximately 12 passages (approximately 7 weeks) in culture, insufficient to derive and analyse stable transfectant clones by drug selection. As this may apply to other primary cells, we describe here how the problem is overcome.
  • Immortalised clones can then be expanded and analysed at will. Where necessary, analysis of transgene expression in the absence of T antigen expression can be achieved by growth at the non-permissive temperature (39°C) . As in examples 3 and 4, once a suitable clone was identified, the cryopreserved sample of non-transformed cells could be thawed and used for nuclear transfer.

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AU739902B2 (en) * 1998-01-16 2001-10-25 Agrobiogen Gmbh Efficient nuclear transfer using fetal fibroblasts
WO1999046982A1 (en) * 1998-03-16 1999-09-23 Relag Pty Ltd Porcine nuclear transfer
EP2199392A3 (de) * 1999-03-04 2010-12-15 Revivicor, Inc. Genetische veränderung von somatischen zellen und deren verwendungen
WO2001088096A2 (en) * 2000-05-15 2001-11-22 Geron Corporation Ovine tissue for xenotransplantation
WO2002009507A1 (de) * 2000-07-27 2002-02-07 Apogene Gmbh & Co. Kg Somatischer klonierungs-gentransfer zur produktion von rekombinanten proteinen, zellen und organen
MXPA03001915A (es) 2000-08-03 2004-09-10 Therapeutic Human Polyclonals Produccion de anticuerpos humanizados en animales transgenicos.
EP1434988A4 (de) 2001-02-23 2005-12-14 Elan Pharm Inc Transgene bace-1-knockouts
AU2003290689A1 (en) 2002-11-08 2004-06-03 Kyowa Hakko Kirin Co., Ltd. Transgenic ungulates having reduced prion protein activity and uses thereof
US7420099B2 (en) 2004-04-22 2008-09-02 Kirin Holdings Kabushiki Kaisha Transgenic animals and uses thereof
SG174053A1 (en) 2006-09-01 2011-09-29 Therapeutic Human Polyclonals Inc Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals
US8557773B2 (en) 2008-05-02 2013-10-15 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Treatment of bleeding with low half-life fibrinogen

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