+

WO1997020035A1 - Creation, maintenance et transfection de cellules souches embryonnaires totipotentes a partir d'embryons d'animaux domestiques - Google Patents

Creation, maintenance et transfection de cellules souches embryonnaires totipotentes a partir d'embryons d'animaux domestiques Download PDF

Info

Publication number
WO1997020035A1
WO1997020035A1 PCT/US1996/018988 US9618988W WO9720035A1 WO 1997020035 A1 WO1997020035 A1 WO 1997020035A1 US 9618988 W US9618988 W US 9618988W WO 9720035 A1 WO9720035 A1 WO 9720035A1
Authority
WO
WIPO (PCT)
Prior art keywords
effective amount
blastocyst
embryonic stem
cells
medium
Prior art date
Application number
PCT/US1996/018988
Other languages
English (en)
Inventor
Kenneth L. White
Irina Polejaeva
John D. Morrey
Original Assignee
Utah State University
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 Utah State University filed Critical Utah State University
Priority to AU12747/97A priority Critical patent/AU1274797A/en
Publication of WO1997020035A1 publication Critical patent/WO1997020035A1/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
    • 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
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/315—Prolactin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/905—Hyaluronic acid
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to animal cell cultures. More particularly, the invention relates to a method of establishing, maintaining, and transfecting totipotent embryonic stem cell lines from domestic animals, such as cattle, sheep, goats, rabbits, and mink.
  • Embryonic stem (ES) cells derived from the inner cell mass (ICM) of developing embryos are totipotent, i.e. have the ability to develop into any type of cell in the body.
  • ICM inner cell mass
  • ES cells typically lose totipotency and begin to differentiate aberrantly into other cell types when propagated in cell culture.
  • Established ES cell lines can be used for rapid propagation of selected domestic animals having valuable or desirable phenotypes.
  • ES cells from embryos from superior animals or transgenic animals can be used to propagate a herd having selected traits within about a year.
  • ES cell lines can also be used to produce transgenic animals at a very high efficiency, up to 100%, whereas the current state-of-the-art with differentiated cells is an efficiency of less than about 5%.
  • Methods of producing ES cell lines to date have been generally unproductive.
  • Several publications describe ES-like cells from livestock, however, very few have reported the production of viable offspring. E. g . , N. Strelchenko, 45 Theriogenology 131-40 (1996) .
  • the domestic animal is preferably a member selected from the group consisting of cattle, sheep, goats, rabbits, and mink.
  • Preferred feeder cells include murine primary embryonic fibroblasts. It is further preferred to pretreat the murine primary embryonic fibroblasts such that mitotic activity thereof is blocked, such as by treatment with mitotnycin C.
  • the medium preferably comprises at least about 10% bovine plasma or bovine plasma that has been fractionated to reduce the amount of mitogens therein. It is also preferred to use bovine plasma from a fetal animal .
  • the medium also preferably comprises an effective amount of leukemia inhibitory factor, hyaluronic acid, alpha- fetoprotein, or mixtures thereof.
  • leukemia inhibitory factor about 1-5 ng/ml of leukemia inhibitory factor, about 10-100 ⁇ g/ml of alpha- fetoprotein, and about 100-4000 ⁇ g/ml of hyaluronic acid are especially preferred ranges.
  • a method of transfecting an undifferentiated, pluripotent embryonic stem cell line of a domestic animal with a selected gene comprises the steps of: (a) obtaining a viable blastocyst, having a zona pellucida, from the animal;
  • a composition for use in establishing an undifferentiated, pluripotent embryonic stem cell line of a domestic animal comprises at least about 10% of bovine plasma and optionally an effective amount of a member selected from the group consisting of leukemia inhibitory factor, hyaluronic acid, alpha-fetoprotein, and mixtures thereof.
  • totipotent refers to cells that have the ability to develop into any type of cell m the body.
  • pluripotent refers to cells that have the ability to develop into any one of several cell types .
  • serum means the liquid portion of blood that is prepared after clotting of the blood, i . e . after the clotting cascade has been carried out and other associated processes, such as release of mitogens, has occurred.
  • plasma means the liquid portion of blood that is prepared without the blood having gone through the clotting cascade. Plasma is prepared from blood that is treated to inhibit occurrence of the clotting cascade. Plasma can also be fractionated, such as by centrifugation, to remove small amounts of mitogens that can be present despite the clotting cascade and associated processes not having occurred. Such mitogens are believed detrimental to establishment and maintenance of ES cell lines Plasma should also preferably be free of platelets.
  • an effective amount means an amount that is nontoxic but sufficient to provide a selected effect and performance
  • an effective amount of mitomycm C is an amount sufficient to block mitotic activity of feeder cells without being toxic thereto
  • An effective amount of a protease is an amount sufficient to digest the zona pellucida without being toxic to the blastocyst.
  • An effective amount of leukemia inhibitory factor or hyaluronic acid is an amount sufficient for inhibiting differentiation of embryonic stem cells without being toxic thereto.
  • an effective amount of alpha-fetoprotein is an amount sufficient to enhance growth of embryonic stem cells without being toxic thereto Further, an effective amount of prolactin is an amount sufficient to reactivate mink blastocysts from embryonic diapause without being toxic thereto. Moreover, an effective amount of a transfection mixture is an amount of lipofection reagent and nucleic acid sufficient to effect transfection of the nucleic acid into embryonic stem cells without being toxic thereto.
  • ES cell lines prepared according to the present invention can be used for propagation of domestic animals by methods that are well known in the art.
  • blastocysts from an undesirable animal can be injected with ES cells prepared from a superior animal or a transgenic animal.
  • the ⁇ e mnected blastocysts result in chimeric animals, some of which can be germ line chimeras that will contain the gene or genes that are desired to be propagated and selected in resulting offspring.
  • nuclear transplantation technology can be used to propagate domestic animal ⁇ having the selected genes and/or traits.
  • Oocytes are collected from an undesirable animal and are enucleated by standard methods
  • the enucleated oocytes are then fused with ES cells from a superior animal or transgenic animal.
  • the fusion results in embryos containing the genome of the superior or transgenic animal.
  • These embryos can then be implanted into suitable recipients for gestation to proceed according to well known methods.
  • G.E. Seidel, Jr. & S.M. Seidel The Embryo Transfer Industry, m New Technologies m Animal Breeding 41-77 (B.G Brackett, G.E.
  • Example 1 The objective of this example is to establish mink ES (MES) cell lines and test them for the presence of alkaline phosphatase (AP) , a biochemical marker characteristic of the earliest developmental stages and also of mouse embryonic stem cells Expanded blastocysts and 4- to 8-cell embryos were flushed from the uteri and oviducts of naturally-mated, wild-type mink. The blastocysts and embryos were then digested with pronase (Sigma Chemical Co., St.
  • MPEF mouse primary embryonic fibroblasts
  • Mitotic activity of MPEF cells was blocked by incubation for 2 hours m 10 ⁇ g/ml mitomycm C (Sigma Chemical Co.) Dulbecco's modified Eagle's medium (DMEM, Hyclone Laboratories, Logan, Utah) supplemented with 10% fetal bovme serum (HyClone Laboratories, Inc.) , 0.1 mM 2- mercaptoethanol (Mallinckrodt Chemical Inc., Chesterfield, MO) , and 0.1 mM non-essential ammo acids (Life Technologies Inc., Gaithersburg, MD) and antibiotics (50-100 IU/ml penicillin, 50 ⁇ g/ml streptomycin; Life Technologies, Inc.) was used for embryo culture.
  • the mink reproductive cycle includes a period of embryonic diapause (delayed implantation of 30-40 days) . Therefore, blastocysts were kept in suspension for 10 days followmg removal from the reproductive tract, during which time the blastocysts remained intact. Then, the blastocysts were "reactivated” by treatment with 5 ⁇ g/ml prolactin (PRL; Sigma Chemical Co. ) . With 24 hours after addition of prolactin, all blastocysts attached to the feeder layers Three MES cell lines were obtained from 4 blastocysts. No MES cell lines were established, however, from 4- to 8-cell embryos.
  • MES cell lines had a high nuclei/cytoplasm ratio, produced simple embryonic bodies, and were positive for AP staining.
  • MES cells differentiated into fibroblast- like and epithelium-like cells when cultured without feeder layers, and these differentiated cells were negative for AP.
  • the MES cell lines were maintained in culture for approximately 5 months without morphological differentiation.
  • MES cells was further investigated. Expanded blastocysts were flushed from the uteri of naturally mated demi minks 13-17 days post-coitum. Pronase was used to remove the zonae pellucidae from embryos, then the embryos were seeded individually into 4-well culture dishes onto feeder layers of mitomycm-C-treated MPEF, as described Example 1
  • the control medium was DMEM supplemented with 10% fetal bovine serum (FBS) , 0.1 M
  • Treatment groups were treated with the control medium further containing the following amendments. (1) 0 ⁇ g/ml PRL, (2) 5 ⁇ g/ml PRL added after 10 days m culture, (3) 10 ng/ml PRL, (4) 1 ⁇ g/ml
  • Bov e ES (BES) cell lines were established and tested for AP according to the procedure of Example 1 except that bovine expanded blastocysts were produced by in vi tro fertilization (IVF) procedures and the prolactin step was omitted.
  • IVF vi tro fertilization
  • Ovaries were collected at a local abattoir and transported to the laboratory in 0.9% saline at a consistent temperature of about 25 C C. The ovaries were then washed thrice in the same solution before aspiration of oocytes.
  • Oocytes were collected by aspirating small antral follicles (1-7 mm diameter) with a disposable 18-gauge needled connected by glass tubing to a 50 ml conical tube, which served as a trap for collection of the oocytes and follicular fluid.
  • H.W. Hawk & R.J. Wall Improved Yields of Bovine Blastocysts from In Vitro-Produced Oocytes.
  • the vacuum pump attached to the trap was set at 150 mm Hg.
  • Maturation medium was TCM- 199 (Morgan, Morton, Parker, 73 Proc. Soc. of Exp. Biol. Med. 1 (1950) ; HyClone Laboratories Catalogue, hereby incorporated by reference) , 10% fetal bovine serum, 0.5 ⁇ g/ml FSH, 5.0 ⁇ g/ml LH.
  • IVM culture was for 24 hours in a humidified 5% carbon dioxide atmosphere at 39°C.
  • Semen used in culture studies met the standard of ⁇ 70% fertilization potential. Straws of frozen semen were thawed in a water bath at 40°C for 1 minute, and the contents of the straw were then transferred to and layered on a 45%-90% Percoll step gradient in a 15 ml
  • the motile spermatozoa were collected and added to the fertilization medium (Fert-TALP, Parrish et al . , supra) to provide a final concentration of 1.0 x
  • HEPES-TALP 1 ml HEPES-TALP supplemented with 0.3% bovine serum albumin. Cumulus and corona cells were removed from the ova by vortexing for 165 seconds. The ova were then placed in 30 ⁇ l drops of culture medium (synthetic oviductal fluid; SOF) , covered with dimethylpolysiloxane, and cultured in vi tro under a humidified 5% oxygen, 90% nitrogen, 5% carbon dioxide atmosphere at 39°C. Embryos that were blastocysts on days 6 and 7 were placed in ES cell culture. Seven BES cell lines were established from 10 blastocysts. All seven of these BES cell lines had a high nuclei/cytoplasm ratio and produced simple embryonic bodies.
  • SOF synthetic oviductal fluid
  • BES cell lines were negative for AP activity, however. BES cell lines spontaneously differentiated into neuron-like, epithelium-like, and fibroblast-like cells when not cultured on feeder layers. BES lines were maintained in culture for approximately 5 months without morphological differentiation. These results suggest that BES cell lines prepared according to the present method are pluripotent, but AP activity appears not to be a useful marker for BES cells.
  • Example 4 ES cells have previously been grown routinely in
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • Example 4 the procedure of Example 4 was followed except that bovine epithelial oviductal cells were used as the feeder layer instead of MPEF cells. None of 27 blastocysts attached to the oviductal cell monolayer. These results show that the selection of cells used as a feeder layer for establishing BES cell lines can have a significant effect thereon. For example, feeder layers of MPEF cells are much superior to bovine epithelial oviductal cells.
  • Example 6 In this example, the procedure of Example 4 was followed except that blastocysts were cultured over MPEF feeder layer in 25% BP, 10% FBS, or 25% FBP supplemented as in Example 4 and additionally with 0 ng/ml, 1 ng/ml, 5 ng/ml, or 10 ng/ml of the cytokine, human leukemia inhibitory factor (LIF) .
  • LIF human leukemia inhibitory factor
  • G.B. Anderson, 3 Animal Biotechnol . 165-176 (1992) has suggested that a mouse culture system and murine LIF are not effective for preventing differentiation of ES cells from domestic animals.
  • the BES lines prepared according to this procedure were maintained in culture for about 7 weeks without morphological differentiation. These results show that about 1-5 ng/ml of human LIF in the culture medium significantly increases the success rate for establishment of BES cell lines.
  • Example 7 In this example, the procedure of Example 4 is followed except that blastocysts are cultured over MPEF feeder layer in 25% BP or 25% FBP supplemented as in Example 4 and additionally with 100-4000 ⁇ g/ml of hyaluronic acid (Sigma Chemical Co. ) .
  • Hyaluronic acid is known to inhibit differentiation and enhance proliferation of cells in culture.
  • S. Kato & T. Miyano Effects of Hyaluronic Acid on the Development of 1- and 2-Cell Porcine Embryos to the Blastocyst Stage in Vitro, 41 Theriogenol. 1299-1305 (1994) (abstract) ; M.T. Longaker et al . , Studies in Fetal Wound Healing: V.
  • Hyaluronic has further been shown to sequester or inactivate transforming growth factor-beta (TGF- ⁇ ) , P. Locci et al . , Transforming Growth Factor Beta (1) -Hyaluronic Acid Interaction, 281 Cell Tissue Res. 317-24 (1995) , which is a differentiation factor in serum or plasma.
  • TGF- ⁇ transforming growth factor-beta
  • P. Locci et al . Transforming Growth Factor Beta (1) -Hyaluronic Acid Interaction, 281 Cell Tissue Res. 317-24 (1995) , which is a differentiation factor in serum or plasma.
  • Hyaluronic acid has also been used to better cultivate porcine embryos, S. Kato & T.
  • Example 8 In this example, the procedure of Example 4 is followed except that blastocysts are cultured over MPEF feeder layer in 25% BP or 25% FBP supplemented as in Example 4 and additionally with about 10-100 ⁇ g/ml of alpha-fetoprotein (Sigma Chemical Co.) .
  • Alpha- fetoprotein is found in body fluids of first trimester fetuses, F. Muller et al., First-trimester Amniotic Fluid Acetylcholinesterase Electrophoresis, 9 Prenatal Diagnosis 173-75 (1989) , which suggests that this protein is important in embryo cultivation.
  • Example 2 efficient transfection of MES cells produced by the procedure of Example 1 was demonstrated. Transfection was with plasmids pGFP-Cl or pGFP-shiva (control) (Clontech, Palo Alto, Calif.) . In pGFP-Cl, the marker gene, green fluorescent protein (GFP) , is under control of the cytomegalovirus immediate early promoter. Three established MES cell lines (passage no. 5) were transfected by lipofection, according to procedures well known in the art, with either the pGFP-Cl or pGFP-shiva plasmids in 35 mm petri dishes. Lipofection reagents were purchased from Life Technologies (cat. # 18292-0xx) .
  • LIPOFECTIN Reagent is a 1:1 (w/w) liposome formulation of the cationic lipid N- [1- (2 , 3-d ⁇ oleyloxy) propyl] -n, n, n -trimethylammonium chloride (DOTMA) , and dioleoyl phosphotidylethanolamme (DOPE) in membrane filtered water LIPOFECTIN Reagent interacts spontaneously with DNA to form a lipid-DNA complex. The fusion of the complex with tissue culture cells results the efficient uptake and expression of the DNA. MES cells were plated the day before transfection and were 60-70% confluent at the time of transfection One microgram of plasmid DNA was diluted 50 ⁇ l of serum-free medium.
  • transfection mixture (3 ⁇ l of lipofection reagent + 47 ⁇ l of serum-free medium) was added to the tube containing plasmid DNA, mixed gently, and incubated for 15 minutes at room temperature. The cells were washed once with 1 ml of serum-free medium and then left 900 ⁇ l of serum-free medium. The lipofection/DNA mixture was added to the dish and gently mixed with the medium After overnight incubation, 1 ml of medium containing 20% FBS was added and the ES cells were incubated for 36 hours. The medium was then replaced with 1 ml of fresh medium containing 10% FBS For selection of stable MES colonies, 200 ⁇ g/ml of G418 (Life Technologies, Inc ) was added to the medium 72 hours after transfection.
  • transfection mixture 3 ⁇ l of lipofection reagent + 47 ⁇ l of serum-free medium
  • the expanded ES cell pool was used for fluorescence detection.
  • ES cells were washed twice with PBS, harvested with the tip of a Pasteur pipet, placed on a slide, and covered with a glass cover slip ES cells were observed using a Nikon Diaphot with lOx or 20x fluorescence objective and a filter set comprising an Omega 405DF40 excitation filter, a 450DRLPO 2 dichroic mirror, and a 510WB40 emission filter.
  • the image was then transmitted via a video camera to a computer loaded with Image-1 software (Universal Imaging) , where images were processed, saved, and displayed on the monitor in pseudocolor.
  • Image-1 software Universal Imaging
  • the intensity of GFP fluorescence in cells containing pGFP-Cl was significantly greater (P( ⁇ .05) by Student's T-test as compared to cells containing pGFP- shiva.
  • Transfection efficiencies in MES transfected with pGFP-shiva were as high as 46%, and in MES transfected with pGFP-Cl about 27%. Very low background fluorescence was detected in untransfected cells.
  • Example 10 In this example, the procedure of Example 1 is followed with the exception that the blastocysts are obtained from a rabbit and the prolactin step is omitted.
  • Example 11 In this example, the procedure of Example 10 is followed with the exception that the blastocysts are obtained from a sheep.
  • Example 12 In this example, the procedure of Example 10 is followed with the exception that the blastocysts are obtained from a goat .

Landscapes

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

Abstract

L'invention concerne un procédé servant à créer des lignées de cellules souches embryonnaires (ES) d'animaux domestiques, tels que du bétail, des moutons, des chèvres, des lapins et des visons. On traite les blastocystes dilatés de l'animal domestique, afin de supprimer la zona pellucida et on les sème sur une couche de cellules nourricières dans une boîte de Pétri contenant un milieu approprié. Ce milieu est composé de plasma à faible teneur en mitogènes à la place de sérum ayant subi la cascade de coagulation. L'apport du facteur inhibiteur de la leucémie (LIF), d'alpha-fetoprotéine et/ou d'acide hyaluronique au milieu améliore également l'efficacité de la création de la lignée de cellules souches. On prépare les lignées de cellules souches embryonnaires de vison au moyen du traitement préalable de blastocystes avec de la prolactine, afin de réactiver les blastocystes depuis la diapause embryonnaire. L'invention concerne également la transfection de ces lignées cellulaires embryonnaires, ainsi qu'une composition servant à créer ces lignées.
PCT/US1996/018988 1995-11-29 1996-11-27 Creation, maintenance et transfection de cellules souches embryonnaires totipotentes a partir d'embryons d'animaux domestiques WO1997020035A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12747/97A AU1274797A (en) 1995-11-29 1996-11-27 Establishment, maintenance, and transfection of totipotent embryonic stem cells from the embryos of domestic animals

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US768995P 1995-11-29 1995-11-29
US60/007,689 1995-11-29

Publications (1)

Publication Number Publication Date
WO1997020035A1 true WO1997020035A1 (fr) 1997-06-05

Family

ID=21727612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/018988 WO1997020035A1 (fr) 1995-11-29 1996-11-27 Creation, maintenance et transfection de cellules souches embryonnaires totipotentes a partir d'embryons d'animaux domestiques

Country Status (2)

Country Link
AU (1) AU1274797A (fr)
WO (1) WO1997020035A1 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039416A1 (fr) * 1997-03-06 1998-09-11 Infigen, Inc. Methode de clonage d'animaux
WO1999009141A1 (fr) * 1997-08-14 1999-02-25 Biotransplant, Inc. Cellules totipotentes porcines et procede de culture a long terme
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
US6090622A (en) * 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
WO2000031237A3 (fr) * 1998-11-24 2000-11-16 Infigen Inc Procede de clonage de porcins
US6245566B1 (en) 1997-03-31 2001-06-12 The Johns Hopkins University School Of Medicine Human embryonic germ cell line and methods of use
US6395958B1 (en) 1997-03-06 2002-05-28 Infigen, Inc. Method of producing a polypeptide in an ungulate
EP1217071A1 (fr) 2000-12-22 2002-06-26 Institut National De La Recherche Agronomique (Inra) Expression position-indépendante et tissus-spécifique d' un transgène dans le lait d'animaux transgéniques
WO2003054169A1 (fr) * 2001-12-21 2003-07-03 Thromb-X Nv Compositions pour la derivation et la culture in vitro de cellules souches embryonnaires avec transmission de lignee germinale
WO2003054170A1 (fr) * 2001-12-21 2003-07-03 Thromb-X Nv Compositions pour la culture et la derivation in vitro de lignees cellulaires de souches embryonnaires avec possibilite de transmission de lignee germinale
WO2003055992A3 (fr) * 2001-12-28 2004-01-15 Cell Therapeutics Scandinavia Methode permettant d'etablir une lignee de cellules souches hematopoietiques multipotentes humaines derivees du blastocyste
US6703209B1 (en) 1998-08-13 2004-03-09 Biotransplant, Inc. Porcine totipotent cells and method for long-term culture
US7504096B1 (en) 1998-07-06 2009-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for in vitro fertilization
US7638328B2 (en) 2003-05-08 2009-12-29 Cellartis Ab Method for efficient transfer of human blastocyst-derived stem cells (hBS cells) from a feeder-supported to a feeder-free culture system, long-term propagation of hBS cells under feeder-free conditions and use of cultured hBS cells for applications in myocardial regeneration
US7700352B2 (en) 2002-10-31 2010-04-20 Riken Composition for culturing mouse pluripotent stem cells
US9617515B2 (en) 2006-02-27 2017-04-11 Moraga Biotechnology Corporation Non-embryonic totipotent blastomere-like stem cells and methods therefor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 14 September 1995, Vol. 214, No. 2, YOSHIDA et al., "Gene Targetting of DT-diaphorase in Mouse Embryonic Stem Cells: Establishment of Null Mutant and Its Mitomycin C-resistance", pages 701-708. *
THERIOGENOLOGY, ANNUAL CONFERENCE OF THE INT., No. 1, WHITE et al., "Effect of Non-serum Supplemented Media on Establishment and Maintenance of Bovine Embryonic Stem-like Cells", page 350. *
THERIOGENOLOGY, ANNUAL CONFERENCE OF THE INTERNATIONAL EMBRYO TRANSFER SOCIETY, 08 January 1995, Vol. 43, No. 1, POLEJAEVAL et al., "Isolation and Long-term Culture of Mink and Bovine Embryonic Stem-like Cells", page 300. *
TRANSGENIC RESEARCH, May 1994, Vol. 3, WOLF et al., "Efficient Generation of Chimaeric Mice Using Embryonic Stem Cells after Long-term Culture in the Presence of Ciliary Neurotrophic Factor", pages 152-158. *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7071373B1 (en) 1993-05-14 2006-07-04 The Board Of Trustees Of The University Of Illinois Transgenic ungulate compositions and methods
US5942435A (en) * 1993-05-14 1999-08-24 The Board Of Trustees Of The University Of Illinois Transgenic swine compositions and methods
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
US6331406B1 (en) * 1997-03-31 2001-12-18 The John Hopkins University School Of Medicine Human enbryonic germ cell and methods of use
US6090622A (en) * 1997-03-31 2000-07-18 The Johns Hopkins School Of Medicine Human embryonic pluripotent germ cells
US6562619B1 (en) 1997-03-31 2003-05-13 The Johns Hopkins University School Of Medicine Differentiation of human embryonic germ cells
US6245566B1 (en) 1997-03-31 2001-06-12 The Johns Hopkins University School Of Medicine Human embryonic germ cell line and methods of use
WO1999009141A1 (fr) * 1997-08-14 1999-02-25 Biotransplant, Inc. Cellules totipotentes porcines et procede de culture a long terme
US7504096B1 (en) 1998-07-06 2009-03-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for in vitro fertilization
US6703209B1 (en) 1998-08-13 2004-03-09 Biotransplant, Inc. Porcine totipotent cells and method for long-term culture
EP1586633A1 (fr) * 1998-11-24 2005-10-19 Infigen, Inc. Méthode de préparation de cellules porcines totipotentes
WO2000031237A3 (fr) * 1998-11-24 2000-11-16 Infigen Inc Procede de clonage de porcins
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
EP1217071A1 (fr) 2000-12-22 2002-06-26 Institut National De La Recherche Agronomique (Inra) Expression position-indépendante et tissus-spécifique d' un transgène dans le lait d'animaux transgéniques
US8993323B2 (en) 2001-12-21 2015-03-31 Thrombogenics N.V. Compositions for the in vitro derivation and culture of embryonic stem (ES) cell lines with germline transmission capability and for the culture of adult stem cells
WO2003054169A1 (fr) * 2001-12-21 2003-07-03 Thromb-X Nv Compositions pour la derivation et la culture in vitro de cellules souches embryonnaires avec transmission de lignee germinale
WO2003054170A1 (fr) * 2001-12-21 2003-07-03 Thromb-X Nv Compositions pour la culture et la derivation in vitro de lignees cellulaires de souches embryonnaires avec possibilite de transmission de lignee germinale
US8697444B2 (en) 2001-12-21 2014-04-15 Thrombogenics N.V. Compositions for the in vitro derivation and culture of embryonic stem (ES) cell lines with germline transmission capability and for the culture of adult stem cells
GB2398795A (en) * 2001-12-28 2004-09-01 Cellartis Ab A method for the establishment of a pluripotent human blastocyst-derived stem cell line
WO2003055992A3 (fr) * 2001-12-28 2004-01-15 Cell Therapeutics Scandinavia Methode permettant d'etablir une lignee de cellules souches hematopoietiques multipotentes humaines derivees du blastocyste
US7700352B2 (en) 2002-10-31 2010-04-20 Riken Composition for culturing mouse pluripotent stem cells
US7638328B2 (en) 2003-05-08 2009-12-29 Cellartis Ab Method for efficient transfer of human blastocyst-derived stem cells (hBS cells) from a feeder-supported to a feeder-free culture system, long-term propagation of hBS cells under feeder-free conditions and use of cultured hBS cells for applications in myocardial regeneration
US9617515B2 (en) 2006-02-27 2017-04-11 Moraga Biotechnology Corporation Non-embryonic totipotent blastomere-like stem cells and methods therefor

Also Published As

Publication number Publication date
AU1274797A (en) 1997-06-19

Similar Documents

Publication Publication Date Title
US6011197A (en) Method of cloning bovines using reprogrammed non-embryonic bovine cells
AU745334B2 (en) Method of cloning animals
AU717529B2 (en) Cultured inner cell mass cell lines derived from ungulate embryos
Ouhibi et al. Co-culture of 1-cell mouse embryos on different cell supports
US20020090722A1 (en) Pluripotent mammalian cells
WO1997020035A1 (fr) Creation, maintenance et transfection de cellules souches embryonnaires totipotentes a partir d'embryons d'animaux domestiques
US6107543A (en) Culture of totipotent embryonic inner cells mass cells and production of bovine animals
CA1335660C (fr) Culture in vitro d'embryons bovins
Goto et al. Co-culture of in vitro fertilized bovine embryos with different cell monolayers
WO1995010599A1 (fr) Cellules analogues a des cellules souches embryonnaires
AU699869B2 (en) EG Cells of Ungulates
US20170088816A1 (en) Mammalian embryonic stem cell isolated from a homogeneous pluripotent outgrowth of a mammalian pre-implantation embryo
Rexroad Jr et al. Development of ovine embryos co-cultured on oviductal cells, embryonic fibroblasts, or STO cell monolayers
US20040093624A1 (en) Method for improving development potential of an embryo and embryos developed therefrom
JP3771500B2 (ja) 無血清培地−無フィーダー細胞系を用いた哺乳動物胚由来未分化細胞の培養及び細胞株樹立方法
EP0855441A2 (fr) Isolement et purification de cellules souches germinales au moyen de particules magnétiques fonctionnant comme des anticorps et méthode pour l'établissement de lignées de cellules souches
US20030134422A1 (en) Stem cell maturation for all tissue lines
CA2474766A1 (fr) Maturation de cellules souches pour tous les types de tissus
AU2001276183B2 (en) A method for improving development potential of an embryo and embryos developed therefrom
AU782358B2 (en) Method of cloning animals
Collodi Strategies for the Isolation, Characterization, and Use of Embryonic Stem Cells
AU2003210566A1 (en) Stem cell maturation for all tissue types
AU7934594A (en) Embryonic stem cell-like cells
AU2001276183A1 (en) A method for improving development potential of an embryo and embryos developed therefrom

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI

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)
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97520650

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载