WO1996007732A1 - Cellules totipotentes pour un transfert nucleaire - Google Patents

Cellules totipotentes pour un transfert nucleaire Download PDF

Info

Publication number
WO1996007732A1
WO1996007732A1 PCT/GB1995/002095 GB9502095W WO9607732A1 WO 1996007732 A1 WO1996007732 A1 WO 1996007732A1 GB 9502095 W GB9502095 W GB 9502095W WO 9607732 A1 WO9607732 A1 WO 9607732A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
tnt
animal
embryo
Prior art date
Application number
PCT/GB1995/002095
Other languages
English (en)
Inventor
James Mcwhir
Keith Henry Stockman Campbell
Original Assignee
Roslin Institute (Edinburgh)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roslin Institute (Edinburgh) filed Critical Roslin Institute (Edinburgh)
Priority to AU33956/95A priority Critical patent/AU3395695A/en
Publication of WO1996007732A1 publication Critical patent/WO1996007732A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8771—Bovine embryos
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • 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

  • This invention relates to the generation of animals, including but not being limited to transgenic animals, and to cells useful in their generation.
  • the cloning and propagation of cells capable of developing into healthy animals are objectives which have been sought for some time by animal breeders and by producers of transgenic animals.
  • Animal breeders dealing with non-transgenic animals have long sought a means of cloning animals of high genetic merit. The nature of such merit will of course depend on the objectives of the breeder, but it is clear that the dairy industry, to take one example, would benefit from the ability to limit births of calves to those of a single sex.
  • transgenesis has been widely used in the mouse to address questions of gene function, and more sparingly in domestic species, in attempts to alter characters with high economic value.
  • whole animal transgenesis in species other than mouse can only be achieved by pronuclear injection or, less commonly, by viral transfection.
  • a method from transgenic cultured cells in the mouse is also available, known as the embryonic stem cell (ES cell) system. This system depends upon the isolation in culture of a specific embryonic lineage which may be modified in vitro while retaining its unique ability to participate in development following transplantation as an intact cell, to early embryos.
  • Proven ES cells are not available in species other than mouse and ES cell nuclei do not support mouse development when transferred to enucleated zygotes or oocytes using present procedures.
  • Pronuclear microinjection is currently the only practicable procedure for gene transfer in ungulates, particularly farm animals.
  • the efficiency of transgenesis in these species suffers due to the granular nature of the cytoplasm which renders it difficult to visualise pronuclei and because of the smaller number of eggs which may be obtained per animal.
  • Additional constraints are the huge cost of animals and the need, in cattle, to transfer single embryos to each recipient female in order to avoid freemartins.
  • mice In contrast to gene transfer, which is much easier in mice than in farm animals, nuclear transfer (NT) in farm animals has been relatively more successful than in the mouse.
  • Calves and lambs have been generated using nuclei from cells of the blastocyst inner cell mass in both cattle (Keefer et al., Biol . Reprod. 50 935-939 (1994) and Sims & First, Proc . Nat 'l . Acad . Sci . USA 90 6143- 6147 (1994)) and sheep (Smith & Wilmut, Biol . Reprod. 40 1027-1035 (1989) ) . Similar experiments in mice are controversial, but have probably not yielded pregnancies.
  • Embryonic stem cells are tissue culture cells isolated from the inner cell mass of the mouse blastocyst which retain in culture their ability to participate in normal development. This capacity is dramatically demonstrated when ES cells are returned to the early embryonic environment, wherein they participate in normal development, giving rise to chimeric animals whose tissues are a mosaic of host embryo and ES cell genotypes. Where ES cells contribute to primordial germ cells, then genetic manipulations to ES cells in vi tro can be passed on to transgenic animals. There has been much interest in the isolation of ES cells from farm animals due, largely, to the expectation that ES nuclei might be competent for nuclear transfer, offering a more efficient route to transgenesis. There is, however, no evidence that competence for NT is a property of ES cells.
  • ES cells are defined by their ability to make germline chimeras.
  • the present invention arises from the idea that an efficient gene transfer system through NT from cultured cells may be achieved without the necessity of isolating ungulate ES lines.
  • a single confluent 25 cm flask of ES cells would generally contain 10 6 to 10 7 cells, and a line would grow to 10 9 to 10 10 cells after five or six passages.
  • the present invention achieves that goal and is based on the discovery that cells derived from the embryonic disc of blastodermic vesicles can be used to establish such lines.
  • an animal cell line derived from an embryonic disc of an ungulate blastodermic vesicle, or the equivalent tissue of an embryo at an equivalent stage in non-ungulate species, cells of which cell line are totipotent for nuclear transfer.
  • TNT cells can be isolated, individually or collectively, and themselves form part of the invention, according to a second aspect of which there are provided isolated animal cells which are derived from an embryonic disc of an ungulate blastodermic vesicle, or the equivalent tissue of an embryo at an equivalent stage in non-ungulate species, and which are totipotent for nuclear transfer.
  • Stages of non-ungulate embryonic development which are equivalent to the blastodermic vesicle in ungulates are those at or immediately after the determination of the three germ layers at gastrulation; examples include the early egg cylinder stage in rodents and the early gastrula in avian species. Tissues from these stages which are equivalent to the ungulate embryonic disc are those which exclude extra-embryonic lineages; examples include embryonic ectoderm plus visceral endoderm in rodents and embryonic disc in avian species. Where, in the relevant species, embryonic ectoderm can be dissected free of endoderm, embryonic ectoderm alone is the preferred tissue.
  • Totipotentiality is the capacity of a cell to differentiate into all cell types; it is a property of both nucleus and cytoplasm.
  • a cell is said to be "totipotent for nuclear transfer" or "TNT" if, following nuclear transfer from that cell to an oocyte, a healthy animal develops to term.
  • Nuclear transfer may be achieved by fusion of the TNT cell with an oocyte, zygote or early (for example, two cell) blastomere.
  • totipotentiality is a property of the TNT nucleus and the recipient cell cytoplasm.
  • any normal (generally enucleated) oocyte, zygote or early blastomere will suffice. Enucleation may be achieved physically, by actual removal of the nucleus, pro-nuclei or metaphase plate (depending on the recipient cell) , or functionally, such as by the application of ultraviolet radiation or another enucleating influence.
  • Cell lines of the invention can be passaged by conventional means and can be kept in permanent culture.
  • permanent culture is meant culture in which significant reproduction of the cells take place and which can be propagated by passaging; the culture is not necessarily kept indefinitely, but can certainly survive for more than ten passages, by which time it would be regarded as permanently established by those skilled in the tissue culture methodology. At that stage, approximately 10 9 to 10 10 or more cells may be present in the culture.
  • the invention is applicable to all animals, including birds such as domestic fowls. In practice, however, it will be to (non-human) mammals, particularly placental mammals, that the greatest commercially useful applicability is presently envisaged.
  • TNT cells and cell lines derived from small mammals such as rabbits and rodents, especially mice and rats, may be useful in some applications, but it is with ungulates, particularly economically important ungulates such as cattle, sheep, goats, water buffalo, camels and pigs that the invention is likely to be most useful, both as a means for cloning animals and as a means for generating transgenic animals.
  • TNT cells from embryos arising from selected matings among elite stock could be used to clone animals of one or more desired genetic characteristics or high genetic merit generally. This would include the capability of limiting births to a single sex, which as previously mentioned is of particular importance in the dairy industry.
  • TNT cells can be genetically manipulated. Then, by a process of nuclear transfer, which in itself is known (see, for example, Campbell et al . , Biol . Reprod. 50 1385-1393 (1994)), transgenic animals may be produced from the genetically altered, cultured cells.
  • the overall procedure is expected to have several advantages -- particularly in the generation of transgenic farm animals over conventional procedures for generating transgenics, namely (1) that fewer recipient animals will be required, (2) that multiple syngeneic founders may be generated using clonal TNT cells, and (3) that the system will permit subtle genetic alteration by gene targeting.
  • transgenic in relation to animals, should not be taken to be limited to referring to animals containing in their germ line one or more genes from another species, although many transgenic animals will contain such a gene or genes. Rather, the term refers more broadly to any animal whose germ line has been the subject of technical intervention by reco binant DNA technology. So, for example, an animal in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic animal for the purposes of this invention as much as an animal to whose germline an exogenous DNA sequence has been added.
  • a process for the preparation of an animal comprising reconstituting an animal embryo by nuclear transfer from a TNT cell as described above, allowing the embryo to develop to term and optionally breeding from the animal so formed.
  • TNT cells may be genetically modified prior to nuclear transfer.
  • micro- injection analogous to injection into the male or female pronucleus of a zygote, may be used as a method of genetic modification, the invention is not limited to that methodology: mass transformation or transfection techniques can also be used.
  • transgenesis for example by transfection with suitable constructs, with or without selectable markers
  • (2a) optionally screen and select for stable integrants - skip for microinjection
  • TNT cells can be isolated from explants of the embryonic disc of animals. More particularly, TNT cells can be isolated from explants of the embryonic discs of early blastodermic vesicles, for example at days 9 and 10 in sheep, and at equivalent stages in other animals. For cattle and pigs, the equivalent stage would be days 11 and 12 of the blastodermic vesicle, and in rodents the equivalent would be days 5 and 6 of the egg cylinder.
  • the most successful procedure for the isolation of TNT cells comprises explanting at least part of the embryonic disc of an animal embryo at the blastodermic vesicle stage in the case of an ungulate, or the equivalent tissue of an embryo at an equivalent stage in non-ungulate species, allowing the development of an ES- like colony of undifferentiated cells until they have acquired an enlarged, more epithelial phenotype (which will usually be by about passage 2) and entering a cell or cells from such a colony into reproductive culture until required.
  • substantially the whole of the embryonic disc, essentially free of trophectoderm, will usually be taken, for example by microdissection or immunosurgery, from the embryo. Explantation will be onto a suitable tissue culture substrate or medium, such as a primary mouse fibroblast feeder layer (or other inactivated feeder) layer in ES medium, or with conditioned or supplemented medium. Approximately 10% of explants have in practice been found to give rise to the ES-like colonies of undifferentiated cells, which by passage 2 have acquire the enlarged, more epithelial phenotype referred to above. Such a colony, or at least one or more of the cells from it, may be entered into permanent culture, as shown for example in Example 1 below. TNT cells from the sheep at passage 3 support development of lambs to term following nuclear transfer to enucleated oocytes (see Example 2 below) .
  • the TNT strategy differs from the ES strategy in that cell lines are isolated from later stage blastodermic vesicles (days 9 and 10 in sheep) and are not required to generate chimeras. TNT cells do not display the classic ES phenotype of small, rounded, undifferentiated cells and are more epithelial in character, growing as a flat monolayer.
  • the TNT strategy of the present invention also differs from that of Sims & First ( Proc . Nat ' l . Acad . Sci . USA 90 6143-6147 (1994)) in that they isolated cells at an earlier stage of development from the inner cell mass of the blastocyst, rather than the embryonic disc of the blastodermic vesicle, and did not appear to have established true cell lines.
  • transgene constructs which may be selectable, by a variety of methods.
  • mass transformation or transfection techniques can be used, including electroporation, viral-mediated transfection and lipofection.
  • the present invention enables investigators to capitalise on events which happen at low frequency: an example of a low frequency event is homologous recombination, which can be harnessed for gene targeting.
  • the transfection or transformation step can be followed by culture in selective medium.
  • the invention is not limited to the use of any particular technique for the introduction of a transgene: the foregoing examples are given merely by way of illustration. Using microinjection, it should be possible to introduce transgenes at passage 2 or 3 without selection. Injected DNA integrates stably at a frequency of about 20% in cultured cells (Lovell-Badge, "Introduction of DNA into embryonic stem cells” In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford, E. J. Robertson, ed. pp 153-182, 1987) .
  • TNT cells preselection of transgenic clones would mean that 100% of liveborn animals would be transgenic, and the only losses would be due to the proportion of TNT-derived blastocysts transferred to final recipients which fail to develop to term.
  • preliminary data suggest that development to blastocyst strongly correlates with development to term (Table 1) , raising the possibility that the numbers of recipients required can be reduced by an order of magnitude using the TNT system.
  • FIGURE 1 shows the appearance of TNT/4 cells at passage 4 (circled) growing on an inactivated fibroblast feeder layer (arrowed) .
  • TNT cells have an enlarged, flattened, epithelial phenotype, contrasting sharply with ES cells, which are small, rounded and have a high nucleus:cytoplasm ratio;
  • FIGURE 2 shows a lamb (left) derived from fusion of a Welsh Mountain-derived TNT cell to a Scottish Blackface oocyte, followed by transfer to a Scottish Blackface foster mother (right) .
  • Lamb and foster mother show characteristic markings of Welsh Mountain (TNT/4 genotype) and Scottish Blackface
  • Embryo culture technique is very similar to that used in attempts at ES isolation, with 2 significant differences: (1) slightly later stage embryos, (blastodermic vesicles) are entered into culture and (2) undifferentiated ES-like early colonies are allowed to differentiate to generate rapidly dividing, permanent epithelial cell lines.
  • loc. ci t . In contrast to Strelchenko et al . , loc. ci t . , and Sims & First, loc . ci t . , who used earlier stages, forty embryonic discs (EDs) at days 9 and 10 were dissected free of trophectoderm and cultured in groups of three to ten on inactivated primary mouse fibroblasts in 24 well culture plates. Culture medium was Glasgow's modified eagles medium (GMEM; Gibco, UK) supplemented with 10% foetal bovine serum (Gibco, UK) and lOOOU/ml of the anti- differentiation agent, leukaemia inhibition factor (LIF) .
  • GMEM Glasgow's modified eagles medium
  • LIF leukaemia inhibition factor
  • EDs were disaggregated by mild trypsinisation after 3-7 days (ie after attachment and evidence of outgrowth) and passaged onto a fresh feeder layer (passage 1) . Within 7-10 days a proportion of wells showed small foci of undifferentiated cells. These were picked and passaged onto fresh feeders again in 24 well plates (passage 2 or P2) . P2 cells expanded rapidly, losing their small, round, undifferentiated phenotype ( Figure 1) . At confluence, P2s were passaged into a single 25cm 2 flask, four such lines were isolated and designated TNT/1 through 4. Three lines were frozen at passage 4. The cell line TNT/4 was thawed and then cultured for a further 6 passages.
  • EXAMPLE 2 Generation of Lambs from TNT Nuclei Recipient oocytes were obtained following superovulation as per donors above, with the exception that no artificial insemination occurred. Unfertilised (Black Welsh Mountain and Scottish Blackface) Mil oocytes were recovered in phosphate buffered saline (PBS) by flushing from the oviduct 31-33 h after gnRH injection. Recovered oocytes were washed in OCM medium (Gibco, UK) and transferred to medium TCM 199 (Gibco, UK) .
  • PBS phosphate buffered saline
  • oocytes were placed in TCM 199 containing 10% FBS, 7.5 ⁇ g/ml cytochalasin B (Sigma, UK) and 5.0 ⁇ g/ml Hoechst 33342 (Sigma, UK) at 37°C for 20 minutes.
  • a small amount of cytoplasm from directly beneath the first polar body was removed with a finely drawn pipette and enucleation was confirmed by exposing the aspirated cytoplast to UV light and checking for the presence of a metaphase plate.
  • Oocytes were activated in a chamber consisting of two parallel platinum electrodes arranged 200 ⁇ m apart in a glass petri dish 9 cm in diameter. Oocytes were placed between the electrodes in 80 ⁇ l of activation medium (0.3 M mannitol, 0.1 mM MgS0 4 , 0.001 mM CaCl 2 ) in distilled water. Activation was induced by a single DC pulse of 1.25 kV/cm for 80 ⁇ s. Embryos were reconstructed by cell fusion (nuclear transfer from TNT cells) between 6 and 12 hours post activation (Campbell et al . , Biol . Reprod. 50 1385-1393 (1994)).
  • Fusion was carried out in the same chamber described above for activation, by application of a single AC pulse of 3V for 5 s followed by 3 DC pulses of 1.25 kV/cm for 80 ⁇ s in activation medium.
  • Reconstructed embryos were cultured in TCM 199 plus 10% FBS and 7.5 ⁇ g cytochalasin B for 1 hour at 37°C in 5% C0 2 , were double embedded in 1% and then 1.2% agar (Difco) and then transferred to the ligated oviduct of unsynchronised Black Welsh Mountain ewes (temporary recipients for in vivo culture) . After 6 days, recipient ewes were killed and the embryos were retrieved by flushing with PBS. Embryos were dissected from the agar. Those which had developed to morula or blastocyst stages were transferred to final synchronised recipients.
  • EXAMPLE 3 Transfection of foreign DNA into TNT cells
  • Foreign DNA has been introduced to TNT cells by: (i) electroporation (ii) by lipofection and (iii) by Ca- phosphate precipitation.
  • Transfection was monitored either by histological staining for marker gene expression or by quantitation of fluorescence catalysed by a luciferase reporter gene in transient assays.
  • the test plasmid used in transient transfections comprised a PGK promoter driving the 3-galactosidase marker.
  • This gene's activity can be detected by staining with the chromogenic substrate 5-bromo-4-chloro-3-indoyl-?-D- galactosidase (X gal; Sigma, Poole, UK) (Bondi et al . , Histochem 76: 155-8 (1982)).
  • X gal 5-bromo-4-chloro-3-indoyl-?-D- galactosidase
  • X gal 5-bromo-4-chloro-3-indoyl-?-D- galactosidase
  • Electroporations were carried out in 800 ⁇ l volumes containing 1 to 2 x 10 6 cells and 1.0 ⁇ g of circular (for transient transfection) or linear (for stable transfection) test plasmid DNA. Electroporations were carried out using a GENE PULSER TM machine (Bio Rad, Herts,
  • Lipofection techniques involve the packaging of DNA into lipid particles whose properties promote fusion with cells membranes. All lipofections were performed in OPTIMEM medium (Life Technologies, Paisley, UK) and LiPOFECTAMiNE (Life Technologies, Paisley, UK) . 2 x 10 s cells were plated onto one 6cm dish for each treatment. Following attachment, medium was replaced with OPTIMEM containing 1.5 ⁇ g DNA and 10 ⁇ g LIPOFECTAMINE, and was incubated for 1-18 hours at which time OPTIMEM was replaced with normal TNT medium (treatment a) .

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur des cellules animales qui sont totipotentes pour un transfert de noyau (cellules TNT) et peuvent être isolées du disque embryonnaire de la vésicule blastodermique d'un ongulé, ou du tissu équivalent d'un embryon à un stade équivalent chez un individu non-ongulé. Il est possible de mettre in vitro ces cellules TNT en culture, ce qui permet d'effectuer un clonage, comme souhaité, et une recombinaison génétique, notamment pour introduire un transgène. Il est possible de transférer les noyaux de ces cellules dans des cellules receveuses et de reconstituer des embryons. De cette manière, on peut cloner des animaux ayant une valeur génétique élevée et produire des animaux transgéniques par des techniques de transformation de masse sur une plus vaste gamme d'espèces que ne le permet la technique de la cellule souche embryonnaire et sans recourir à la micro-injection pronucléaire.
PCT/GB1995/002095 1994-09-05 1995-09-05 Cellules totipotentes pour un transfert nucleaire WO1996007732A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU33956/95A AU3395695A (en) 1994-09-05 1995-09-05 Totipotent cells for nuclear transfer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9417831.6 1994-09-05
GB9417831A GB9417831D0 (en) 1994-09-05 1994-09-05 Biological manipulation

Publications (1)

Publication Number Publication Date
WO1996007732A1 true WO1996007732A1 (fr) 1996-03-14

Family

ID=10760840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1995/002095 WO1996007732A1 (fr) 1994-09-05 1995-09-05 Cellules totipotentes pour un transfert nucleaire

Country Status (3)

Country Link
AU (1) AU3395695A (fr)
GB (1) GB9417831D0 (fr)
WO (1) WO1996007732A1 (fr)

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
WO1998030683A2 (fr) * 1997-01-10 1998-07-16 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
WO1998037183A1 (fr) * 1997-02-20 1998-08-27 Ppl Therapeutics (Scotland) Limited Production de cellules donneuses transgeniques pour le transfert nucleaire
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
GB2331751A (en) * 1995-08-31 1999-06-02 Roslin Inst Edinburgh Quiescent cell populations for nuclear transfer
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
WO2000031237A2 (fr) * 1998-11-24 2000-06-02 Infigen, Inc. Procede de clonage de porcins
US6235969B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts Cloning pigs using donor nuclei from non-quiescent differentiated cells
US6252133B1 (en) 1995-08-31 2001-06-26 Roslin Institute (Edinburgh) Unactivated oocytes as cytoplast recipients of quiescent and non-quiescent cell nuclei, while maintaining correct ploidy
US6331659B1 (en) 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
US6576464B2 (en) 2000-11-27 2003-06-10 Geron Corporation Methods for providing differentiated stem cells
US6700037B2 (en) 1998-11-24 2004-03-02 Infigen, Inc. Method of cloning porcine animals
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
US7304204B2 (en) 1995-08-31 2007-12-04 Roslin Institute Ungulates produced by nuclear transfer of G1 cells
US7361804B1 (en) 1997-02-19 2008-04-22 Roslin Institute (Edinburgh) Unactivated oocytes in nuclear transfer to produce ungulates
WO2008121199A2 (fr) 2007-03-28 2008-10-09 University Of Iowa Research Foundation Modèles d'animaux transgéniques pour une maladie
EP2138583A1 (fr) 2000-12-22 2009-12-30 Institut National De La Recherche Agronomique Expression position-indépendante et tissus-spécifique d' un transgène dans le lait d'animaux transgéniques
WO2011133889A2 (fr) 2010-04-23 2011-10-27 Cold Spring Harbor Laboratory Sharn présentant une nouvelle conception structurelle
WO2013067328A1 (fr) 2011-11-03 2013-05-10 University Of Iowa Research Foundation Modèles de la mucoviscidose sur cochons transgéniques
US8618352B2 (en) 2007-03-28 2013-12-31 University Of Iowa Research Foundation Transgenic porcine models of cystic fibrosis
US8912386B2 (en) 2007-03-28 2014-12-16 University Of Iowa Research Foundation Transgenic pig model of cystic fibrosis
EP3000877A1 (fr) 2004-06-09 2016-03-30 The University Court of the University of Edinburgh Cellules souches neurales
US9820475B2 (en) 2011-05-16 2017-11-21 The Curators Of The University Of Missouri Porcine reproductive and respiratory syndrome virus resistant animals
US10091975B2 (en) 2015-08-06 2018-10-09 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US11160260B2 (en) 2018-04-17 2021-11-02 The Curators Of The University Of Missouri Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV)
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026884A1 (fr) * 1993-05-14 1994-11-24 Biotechnology Research And Development Corporation Cellules souches embryonnaires destinees a la reproduction d'ongules chimeres et transgeniques
WO1995016770A1 (fr) * 1993-12-17 1995-06-22 Abs Global, Inc. Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026884A1 (fr) * 1993-05-14 1994-11-24 Biotechnology Research And Development Corporation Cellules souches embryonnaires destinees a la reproduction d'ongules chimeres et transgeniques
WO1995016770A1 (fr) * 1993-12-17 1995-06-22 Abs Global, Inc. Cellules souches embryonnaires derivees de cellules du stade preblastocyste d'ongule et leur utilisation pour produire des ongules chimeres et transgeniques clones

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CAMPBELL K ET AL: "Production of live lambs following nuclear transfer of cultured embryonic disc cells.", ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, CALGARY, ALBERTA, CANADA, JANUARY 8-10, 1995. THERIOGENOLOGY 43 (1). 1995. 181. *
CAMPBELL K.H.S. ET AL.: "Improved Development to Blastocyst of Ovine Nuclear Transfer Embryos Reconstructed during the Presumptive S-Phase of Endonucleated Activated Oocytes", BIOLOGY OF REPRODUCTION, vol. 50, no. 6, pages 1385 - 1393 *
FIRST N L ET AL: "Systems for Production of Calves from Cultured Bovine Embryonic Cells.", REPRODUCTION FERTILITY AND DEVELOPMENT 6 (5). 1994. 553-562. *
KEEFER C.I.: "Bovine Inner Cell Mass Cells as Donor Nuclei in the Production of Nuclear Transfer Embryos and Calves", BIOLOGY OF REPRODUCTION, vol. 50, no. 4, pages 935 - 939 *
SIMS, MICHELLE ET AL: "Production of calves by transfer of nuclei from cultured inner cell mass cells", PROC. NATL. ACAD. SCI. U. S. A. (1994), 91(13), 6143-7 *
STICE S ET AL: "Bovine pluripotent embryonic cells contribute to nuclear transfer and chimeric fetuses.", ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, MELBOURNE, VICTORIA, AUSTRALIA, JANUARY 9-11, 1994. THERIOGENOLOGY 41 (1). 1994. 301. *
STRELCHENKO N ET AL: "Bovine embryonic pluripotent cell lines derived from morula stage embryos.", ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, MELBOURNE, VICTORIA, AUSTRALIA, JANUARY 9-11, 1994. THERIOGENOLOGY 41 (1). 1994. 304. *
STRELCHENKO N ET AL: "Differentiation of bovine pluripotential stem cells in vitro.", EXPERIMENTAL BIOLOGY 95, PART II, ATLANTA, GEORGIA, USA, APRIL 9-13, 1995. FASEB JOURNAL 9 (4). 1995. A833. *

Cited By (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
US7071373B1 (en) 1993-05-14 2006-07-04 The Board Of Trustees Of The University Of Illinois Transgenic ungulate compositions and methods
US7329796B2 (en) 1995-08-31 2008-02-12 Roslin Institute (Edinburgh) Serial nuclear transfer of ungulate embryos
GB2331751A (en) * 1995-08-31 1999-06-02 Roslin Inst Edinburgh Quiescent cell populations for nuclear transfer
US6147276A (en) * 1995-08-31 2000-11-14 Roslin Institute (Edinburgh) Quiescent cell populations for nuclear transfer in the production of non-human mammals and non-human mammalian embryos
US7355094B2 (en) 1995-08-31 2008-04-08 Roslin Institute (Edinburgh) Methods of ungulate nuclear transfer
US7326825B2 (en) 1995-08-31 2008-02-05 Roslin Institute (Edinburgh) Methods of ungulate nuclear transfer
EP0930009A1 (fr) * 1995-08-31 1999-07-21 Roslin Institute (Edinburgh) Population de cellules quiescentes pour transfert nucléaire
US7524677B2 (en) 1995-08-31 2009-04-28 Rosiin Institute (Edinburgh) Mammalian cultured inner cell mass cell culture using a g1 cell as nuclear donor
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
GB2331751B (en) * 1995-08-31 2000-01-19 Roslin Inst Quiescent cell populations for nuclear transfer
GB2318578B (en) * 1995-08-31 2000-01-19 Roslin Inst Quiescent cell populations for nuclear transfer
GB2318578A (en) * 1995-08-31 1998-04-29 Roslin Inst Quiescent cell populations for nuclear transfer
EP1005789A2 (fr) * 1995-08-31 2000-06-07 Roslin Institute (Edinburgh) Population de cellules quiescentes pour transfert nucléaire
US7432415B2 (en) 1995-08-31 2008-10-07 Roslin Institute (Edinburgh) Unactivated oocytes as cytoplast recipients for nonprimate mammalian and pig nuclear transfer
US7514258B2 (en) 1995-08-31 2009-04-07 Roslin Institute (Edinburgh) Mammalian cultured inner cell mass cell culture using a quiescent cell as nuclear
US7332648B2 (en) 1995-08-31 2008-02-19 Roslin Institute Unactivated oocytes in nuclear transfer to produce cultured inner cell mass cells and ungulates
US7326824B2 (en) 1995-08-31 2008-02-05 Roslin Institute (Edinburgh) Unactivated ungulate oocytes to produce a transgenic ungulate by nuclear transfer
US6252133B1 (en) 1995-08-31 2001-06-26 Roslin Institute (Edinburgh) Unactivated oocytes as cytoplast recipients of quiescent and non-quiescent cell nuclei, while maintaining correct ploidy
US7321075B2 (en) 1995-08-31 2008-01-22 Roslin Institute (Edinburgh) Serial nuclear transfer of ungulate embryos
US7321076B2 (en) 1995-08-31 2008-01-22 Roslin Institute Unactivated ungulate oocytes to produce a cloned ungulate by nuclear transfer
KR100793220B1 (ko) * 1995-08-31 2008-01-10 로슬린 인스티튜트(에딘버러) 핵이식용의 휴지기 세포 모집단
US6525243B1 (en) 1995-08-31 2003-02-25 Roslin Institute Unactivated oocytes as cytoplast recipients of quiescent cell nuclei while maintaining correct ploidy
US7307198B2 (en) 1995-08-31 2007-12-11 Roslin Institute Ungulates produced by nuclear transfer of G1 cells
US7304204B2 (en) 1995-08-31 2007-12-04 Roslin Institute Ungulates produced by nuclear transfer of G1 cells
US7232938B2 (en) 1995-08-31 2007-06-19 Roslin Institute Cloning ungulates from a quiescent donor cell
EP1005789A3 (fr) * 1995-08-31 2005-03-02 Roslin Institute (Edinburgh) Population de cellules quiescentes pour transfert nucléaire
US6235970B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts, Amherst Campus CICM cells and non-human mammalian embryos prepared by nuclear transfer of a proliferating differentiated cell or its nucleus
US5945577A (en) * 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
CN1248288B (zh) * 1997-01-10 2011-11-09 马萨诸塞大学，马萨诸塞州高等教育公立研究所 用分化胎儿和成年供体细胞进行的核转移
EP1808484A1 (fr) * 1997-01-10 2007-07-18 University of Massachusetts, a Public Institution of Higher Education of The Commonwealth of Massachusetts, Transfert nucléaire avec cellules donatrices adultes et fýtales différenciées
WO1998030683A2 (fr) * 1997-01-10 1998-07-16 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
WO1998030683A3 (fr) * 1997-01-10 1998-09-17 Univ Massachusetts Transfert nucleaire au moyen de cellules donneuses foetales et adultes differentiees
US6235969B1 (en) * 1997-01-10 2001-05-22 University Of Massachusetts Cloning pigs using donor nuclei from non-quiescent differentiated cells
US7361804B1 (en) 1997-02-19 2008-04-22 Roslin Institute (Edinburgh) Unactivated oocytes in nuclear transfer to produce ungulates
WO1998037183A1 (fr) * 1997-02-20 1998-08-27 Ppl Therapeutics (Scotland) Limited Production de cellules donneuses transgeniques pour le transfert nucleaire
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
US6603059B1 (en) 1997-03-06 2003-08-05 Infigen, Inc. Method of cloning animals
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
US6331659B1 (en) 1998-01-21 2001-12-18 University Of Hawaii Cumulus cells as nuclear donors
WO2000031237A2 (fr) * 1998-11-24 2000-06-02 Infigen, Inc. Procede de clonage de porcins
WO2000031237A3 (fr) * 1998-11-24 2000-11-16 Infigen Inc Procede de clonage de porcins
US6258998B1 (en) 1998-11-24 2001-07-10 Infigen, Inc. Method of cloning porcine animals
US6700037B2 (en) 1998-11-24 2004-03-02 Infigen, Inc. Method of cloning porcine animals
EP1586633A1 (fr) * 1998-11-24 2005-10-19 Infigen, Inc. Méthode de préparation de cellules porcines totipotentes
US7547818B2 (en) 2000-03-15 2009-06-16 The University Of Georgia Research Foundation, Inc. Metaphase donor cells for effective nuclear reprogramming in mammals
US6906238B2 (en) 2000-03-15 2005-06-14 University Of Georgia Research Foundation, Inc. Effective nuclear reprogramming in mammals using CDK2 inhibitors
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
US6576464B2 (en) 2000-11-27 2003-06-10 Geron Corporation Methods for providing differentiated stem cells
US8426198B2 (en) 2000-11-27 2013-04-23 Geron Corporation In vitro differentiated cell and human embryonic stem cell population
US9023645B2 (en) 2000-11-27 2015-05-05 Asterias Biotherapeutics, Inc. Isolated in vitro cell population comprising primate pluripotent stem cells containing a nucleic acid construct and differentiated progeny of the pluripotent stem cells
EP2138583A1 (fr) 2000-12-22 2009-12-30 Institut National De La Recherche Agronomique Expression position-indépendante et tissus-spécifique d' un transgène dans le lait d'animaux transgéniques
EP3000877A1 (fr) 2004-06-09 2016-03-30 The University Court of the University of Edinburgh Cellules souches neurales
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals
US7989675B2 (en) 2007-03-28 2011-08-02 University Of Iowa Research Foundation Method of identifying compounds using a transgenic pig model of cystic fibrosis
WO2008121199A2 (fr) 2007-03-28 2008-10-09 University Of Iowa Research Foundation Modèles d'animaux transgéniques pour une maladie
US8618352B2 (en) 2007-03-28 2013-12-31 University Of Iowa Research Foundation Transgenic porcine models of cystic fibrosis
US8912386B2 (en) 2007-03-28 2014-12-16 University Of Iowa Research Foundation Transgenic pig model of cystic fibrosis
WO2011133889A2 (fr) 2010-04-23 2011-10-27 Cold Spring Harbor Laboratory Sharn présentant une nouvelle conception structurelle
EP3502254A1 (fr) 2010-04-23 2019-06-26 Cold Spring Harbor Laboratory Nouveaux arnsh structurellement conçus
US9820475B2 (en) 2011-05-16 2017-11-21 The Curators Of The University Of Missouri Porcine reproductive and respiratory syndrome virus resistant animals
US10080353B2 (en) 2011-05-16 2018-09-25 The Curators Of The University Of Missouri Porcine reproductive and respiratory syndrome virus resistant animals
EP3420813A1 (fr) 2011-05-16 2019-01-02 The Curators Of The University Of Missouri Animaux résistants au virus du syndrome reproducteur et respiratoire porcin
US10405526B2 (en) 2011-05-16 2019-09-10 The Curators Of The University Of Missouri Porcine reproductive and respiratory syndrome virus resistant animals
US11019809B2 (en) 2011-05-16 2021-06-01 The Curators Of The University Of Missouri Porcine reproductive and respiratory syndrome virus resistant animals
WO2013067328A1 (fr) 2011-11-03 2013-05-10 University Of Iowa Research Foundation Modèles de la mucoviscidose sur cochons transgéniques
US10091975B2 (en) 2015-08-06 2018-10-09 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US10827730B2 (en) 2015-08-06 2020-11-10 The Curators Of The University Of Missouri Pathogen-resistant animals having modified CD163 genes
US11160260B2 (en) 2018-04-17 2021-11-02 The Curators Of The University Of Missouri Methods for protecting porcine fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV)

Also Published As

Publication number Publication date
GB9417831D0 (en) 1994-10-26
AU3395695A (en) 1996-03-27

Similar Documents

Publication Publication Date Title
WO1996007732A1 (fr) Cellules totipotentes pour un transfert nucleaire
US5994619A (en) Production of chimeric bovine or porcine animals using cultured inner cell mass cells
Cibelli et al. Trasgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells
JP2019030310A (ja) ｉＰＳ細胞とＢＬＡＳＴＯＣＹＳＴ ＣＯＭＰＬＥＭＥＮＴＡＴＩＯＮを利用した臓器再生法
KR20180091821A (ko) 유전적 상보성에 의한 인간화 car t-세포 및 혈소판의 조작방법
JP2011188873A (ja) 体細胞の遺伝的改変とそれらの使用
JP2007252387A (ja) 動物トランスジェニック幹細胞の単離、選択、および増殖
US7598082B1 (en) Process of mammalian cell reprogramming through production of a heterokaryon
JP4095898B2 (ja) 人工染色体を含むトランスジェニック動物のクローニング
US20100122356A1 (en) Pig model for psoriasis
Moens et al. Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells
JP4845073B2 (ja) 再構築受精卵の作製方法及びそれを用いたトランスジェニック胚の作製方法
CA2396210A1 (fr) Procede de clonage d'animaux avec modifications genetiques ciblees, par transfert de noyaux cellulaires somatiques males ou femelles a culture a long terme, comprenant des modifications genetiques provoquees artificiellement, dans des cellules receveuses enuclees
JP2005515782A6 (ja) 再構築された胚へ核を移植した後の融合および活性化のための方法およびシステム
Yabuuchi et al. Effects of nuclear transfer procedures on ES cell cloning efficiency in the mouse
CA2336437A1 (fr) Reprogrammation cellulaire par production d'heterocaryon
US20060021070A1 (en) Production of chimeric bovine or porcine animals using cultured inner cell mass
EP3120700A1 (fr) Microinjection dans le noyau d'une cellule après transfert nucléaire de cellules somatiques
KR20020008398A (ko) 핵 이식용 세포분열 말기의 세포핵 결실 난모세포
AU771102B2 (en) Cell reprogramming
Funahashi et al. Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle
US20040077077A1 (en) Novel methods for the production of cloned mammals, mammals cloned according to the methods, and methods of use of same
An et al. Cloned calves produced by nuclear transfer from cultured cumulus cells
Meng et al. Cloning of rabbits
Pinkert Genetic engineering of farm mammals

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AU BB BG BR BY CA CN CZ EE FI GE HU IS JP KG KP KR KZ MD MG MK MN MX NO NZ PL RO RU SD SG SI SK TJ TM TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase