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WO2001019181A1 - Clonage de porcs a l'aide de cellules ou de noyaux de donneurs provenant de cellules differenciees et production de porcins multipotents - Google Patents

Clonage de porcs a l'aide de cellules ou de noyaux de donneurs provenant de cellules differenciees et production de porcins multipotents Download PDF

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Publication number
WO2001019181A1
WO2001019181A1 PCT/US2000/024958 US0024958W WO0119181A1 WO 2001019181 A1 WO2001019181 A1 WO 2001019181A1 US 0024958 W US0024958 W US 0024958W WO 0119181 A1 WO0119181 A1 WO 0119181A1
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WO
WIPO (PCT)
Prior art keywords
cell
cells
pig
differentiated
nucleus
Prior art date
Application number
PCT/US2000/024958
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English (en)
Inventor
Steven L. Stice
Jose Cibelli
James Robl
Paul Golueke
Original Assignee
University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusets, As Represented By Its Amherst Campus
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
Priority claimed from US09/394,902 external-priority patent/US7291764B1/en
Application filed by University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusets, As Represented By Its Amherst Campus filed Critical University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusets, As Represented By Its Amherst Campus
Priority to BR0013959-9A priority Critical patent/BR0013959A/pt
Priority to JP2001522835A priority patent/JP2003509031A/ja
Priority to EP00961827A priority patent/EP1241931A4/fr
Priority to CA002381124A priority patent/CA2381124A1/fr
Priority to IL14850800A priority patent/IL148508A0/xx
Priority to AU73725/00A priority patent/AU7372500A/en
Priority to MXPA02002653A priority patent/MXPA02002653A/es
Publication of WO2001019181A1 publication Critical patent/WO2001019181A1/fr

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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
    • 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 cloning procedures in which cell nuclei derived
  • the nuclei are reprogrammed to direct the development of cloned embryos
  • CICM pluripotent cultured inner cell mass cells
  • ICM inner cell mass
  • bovine pluripotent embryonic cells in nuclear transfer and the production of bovine pluripotent embryonic cells
  • embryonic cells adult cells could be used in a bovine cloning procedure to produce embryos.
  • Cloning pig cells is more difficult in comparison with cells of other species. This
  • heterologous DNA is introduced into either early embryos or embryonic cell
  • mice cannot be easily accomplished with early embryo transgenic procedures. Embryonic stem cells in mice have enabled researchers to select for transgenic
  • embryonic cell lines must be maintained in an undifferentiated state that requires feeder
  • preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-
  • ES cells can be
  • Mouse ES cells can give rise to germline chimeras when introduced into
  • animals e.g., ungulates
  • nuclei from like preimplantation livestock embryos support the
  • embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned
  • bovine embryonic stem cell-like cell lines which survived three passages, but were lost
  • embryoid bodies and spontaneously differentiated into at least two different cell types.
  • HES1 a pattern of homeobox genes which is believed to be expressed by ES cells
  • STO mouse fibroblast feeder cells
  • charcoal-stripped serum (rather than normal serum) to supplement the
  • epithelial cells more than pluripotent ICM cells.
  • such differentiated cells will comprise
  • pigs which involves transplantation of a differentiated pig cell or nucleus thereof into an
  • NT units genetically engineered or transgenic pigs (i.e., NT units, fetuses, offspring).
  • invention also provides genetically engineered or transgenic pigs, including those made
  • the invention also provides a differentiated cell or cell nucleus for formation of a NT unit.
  • the invention also provides
  • CICM cells which involves transplantation of a nucleus of a differentiated pig cell or such
  • the invention also provides
  • pluripotent pig CICM cells and cell lines produced by such a method pluripotent pig CICM cells and cell lines produced by such a method.
  • CICM cells may be used within the same species or
  • injuries include Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries,
  • multiple sclerosis muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, burns, vascular diseases, urinary tract diseases, as well as for the treatment
  • tissues may be used within the same species or across species.
  • NT units, fetuses or offspring, or pig CICM cells produced according to the invention in
  • vitro e.g. for study of cell differentiation and for assay purposes, e.g. for drug studies.
  • pig CICMs can be introduced into SCID mice.
  • Such therapies include by way of example
  • fetuses or offspring or pig CICM cells produced according to the invention, or transgenic
  • offspring produced according to the invention in order to produce pharmacologically
  • the present invention provides a method for cloning a pig
  • the method comprises:
  • blastomere which is optionally enucleated under conditions suitable for the formation
  • NT nuclear transfer
  • the activated nuclear transfer unit is cultured until greater than the 2-
  • the culture medium will comprise known substituents, e.g.,
  • hormones, salts, that promote NT embryo development may further optionally be
  • the host pig will optionally comprise "helper embryos", e.g., normal pig
  • helper embryos will preferably number from two to one
  • the cells, tissues and/or organs of the fetus are advantageously used in the area of
  • the present invention also includes a method of cloning a genetically engineered
  • transgenic pig by which a desired DNA sequence is inserted, removed or modified in
  • engineered or transgenic pigs produced by such a method are advantageously used in the area of cell, tissue and/or organ transplantation, production of desirable genotypes, and
  • cloned fetus, embryo, or offspring is to be used to produce cells, tissues or
  • carbohydrate epitopes are involved in rejection responses, i.e., Gal ⁇ l-Gal on the vascular endothelial
  • epitopes or replace such epitopes (e.g., mask) with other carbohydrate epitopes
  • the ⁇ Gal epitopes may be removed enzymatically in vivo by
  • MHC major histo-compatibility complex
  • beta 2-microglobulin a peptide that forms part of the
  • TAP-1 and/or TAP-2 TAP-1 and/or TAP-2
  • TAP-2 transport the peptide fragments across the membrane of the endoplasmic
  • inhibitory cytokines such as LL-4, soluble CTLA-4, CTLA4-Ig, anti-CD40,
  • CD40-L CD 154
  • other inhibitors of receptor-ligand pairs or Fas ligand may inhibit
  • rejection e.g., by inducing tolerance to the transplanted xenograft. Also, rejection may
  • hemeoxygenase (HO-1) can potentiate xenograft survival. Also, hemeoxygenase, (HO-1) can potentiate xenograft survival. Also, hemeoxygenase, (HO-1) can potentiate xenograft survival. Also, hemeoxygenase, (HO-1) can potentiate xenograft survival. Also, hemeoxygenase, (HO-1) can potentiate xenograft survival. Also,
  • anti-apoptotic genes e.g, which inhibit transcriptional activation can be over-expressed
  • endogenous porcine retroviruses may be eliminated to prevent the risk
  • this pathway may be inhibited by genetic modification. Specifically, the
  • DAF Decay Accelerating Factor
  • MCP Membrane Cofactor Protein
  • CD46 CD46
  • CD59 CD59
  • human complement-inhibiting proteins e.g., DAF, MCP
  • the cells, tissues or organs may be cultured in vitro in the presence of
  • donor cells and other agents e.g., CTLA-4 Ig, immunotoxins, anti-CD40-L, prior to
  • transplantation which include by way of example cyclosporine, glucocorticoids, FK-506,
  • the present invention provides a method for producing pig
  • CICM plural cells.
  • the method comprises:
  • blastomere optionally enucleated, under conditions suitable for the formation of a
  • the activated nuclear transfer unit is cultured until greater than the 2-
  • the resultant pig CICM cells are advantageously used in the
  • the present invention provides improved procedures for cloning pigs by nuclear
  • nuclear transfer or nuclear transplantation In the subject application, nuclear transfer or nuclear
  • transplantation or NT are used interchangeably.
  • cell nuclei derived from differentiated pig cells are provided.
  • embryos can also be combined with fertilized embryos to produce chimeric embryos,
  • a feeder layer to prevent overt differentiation of the donor cell to be used in the cloning
  • the present invention uses differentiated cells.
  • the present invention also allows simplification of transgenic procedures by
  • these cells can be clonally propagated without cytokines, conditioned media and/or feeder
  • transgenic pig embryos are used in cloning procedures according to the invention, transgenic pig embryos
  • embryos can be used to produce CICM cell lines or other embryonic cell lines.
  • the present invention eliminates the need to derive and maintain in vitro an
  • desired differentiated cells will be genetically modified, preferably in
  • tissue culture these genetically modified cells used to produce a cloned fetus or animal,
  • differentiated cells are then derived from the cloned fetus or animal, subjected to an
  • Transgenic CICM cells produced according to the invention can be maintained
  • these CICM's will be maintained in an undifferentiated state according to
  • the present invention can also be used to produce cloned pig fetuses, offspring or
  • CICM cells which can be used, for example, in cell, tissue and organ transplantation.
  • This process can provide a source of "materials" for many medical and
  • veterinary therapies including cell and gene therapy. If the cells are transferred back into
  • hematopoietic chimerism can be used to avoid immunological rejection among
  • the present invention provides a method for cloning a pig.
  • the pig will be produced by a nuclear transfer process comprising the following
  • enucleated oocyte or blastomere e.g., by fusion or injection, to form NT units;
  • the activated nuclear transfer unit is cultured until greater than the 2-
  • the host pig will contain one or more "helper"
  • embryos e.g., normal pig embryos, tetraploid embryos, or parthenogenetic embryos to
  • the present invention also includes a method of cloning a genetically engineered
  • transgenic pig by which a desired DNA sequence is inserted, removed or modified in
  • cloned pigs obtained according to the
  • these clones will comprise the identical genotype as a previously existing
  • pigs according to the invention can be used to produced a desired protein, such as a
  • That desired protein can then be isolated from the
  • sequence may confer an agriculturally useful trait to the transgenic pig, such as disease
  • the exogenous DNA may encode one or more
  • DNAs that inhibit rejection of such cells in a heterologous host e.g., human.
  • a heterologous host e.g., human.
  • the pig will express one or more human genes, e.g.,
  • porcine factor VIII if human factor VIII is expressed
  • the present invention further provides for the use of NT fetuses and NT and
  • the present invention provides a method for producing pig
  • the method comprises:
  • nuclear transfer unit is cultured until greater than the 2-cell developmental stage.
  • the pig CICM cells are advantageously used in the area of cell, tissue and organ
  • a fetus is the unborn young of a viviparous animal after it has
  • a mammal is an adult from birth until death.
  • the NT units will be cultured to a size of at least 2 to 400 cells,
  • Nuclear transfer techniques or nuclear transplantation techniques are known in the
  • Differentiated pig cells are those cells
  • the differentiated cells are those which are past the early embryonic stage. More particularly, the differentiated cells are
  • differentiated cells may be derived from ectoderm, mesoderm or endoderm.
  • the differentiated cell will be an active proliferating
  • non-quiescent cell i.e., in G G 2 or M cell phase.
  • Such cells may be obtained directly
  • such differentiating cells may be derived from a non-porcine animal, e.g., a SCID mouse,
  • Suitable differentiated cells useful as the
  • donor cell or nuclei include somatic and germ cells, and nuclei derived therefrom.
  • Pig cells may be obtained by well known methods. Pig cells useful in the present
  • inventions include, by way of example, epithelial cells, cumulus cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes,
  • lymphocytes B and T lymphocytes
  • erythrocytes erythrocytes
  • dendritic cells dendritic cells
  • macrophages macrophages
  • monocytes monocytes, mononuclear cells, and other immune cells, fibroblasts, cardiac muscle cells,
  • pig cells used for nuclear transfer may be any suitable pig cells used for nuclear transfer.
  • the pig cells used for nuclear transfer may be any suitable pig cells used for nuclear transfer.
  • organs e.g., skin, lung, pancreas, liver, stomach, intestine, heart,
  • stem cells reproductive organs, bladder, kidney, urethra and other urinary organs, etc. Also, stem cells
  • cells for specific differentiated cell types may be useful donor cells, e.g., hematopoietic
  • Suitable donor cells i.e.,
  • cells useful in the subject invention may be obtained from any cell or organ of the body.
  • donor cells are intended to include both somatic and germ cells.
  • Fibroblast cells are an ideal cell type because they can be obtained from
  • Fibroblast cells are differentiated
  • the present invention is novel because differentiated cell types are used.
  • the present invention is novel because differentiated cell types are used.
  • invention is advantageous because these cells can be easily propagated, genetically
  • oocytes can be matured in vitro before these cells are used as recipient cells
  • oocytes from pig ovaries e.g., pig ovaries obtained at a slaughterhouse, and maturing the
  • the “maturation period” As used herein for calculation of time periods,
  • aspiration refers to aspiration of the immature oocyte from ovarian follicles.
  • metaphase II stage oocytes which have been matured in vivo have
  • hCG chorionic gonadotropin
  • the oocyte may be matured in vitro after fusion.
  • the oocyte may be matured in vitro after fusion.
  • oocyte as the recipient oocyte because at this stage it is believed that the oocyte can be
  • the oocyte activation period generally ranges from about 16-52
  • immature oocytes can be matured in vitro in suitable maturation
  • porcine oocytes are matured in vitro, e.g.,
  • HCG/PMSG HCG/PMSG and cAMP, which are then washed with HECM/HEPES and
  • sucrose preferably three times, and then placed for about 20 hours in same NCSU 37
  • Matured oocytes, wherein maturation may be effected in vitro (e.g., as described
  • oocytes may be matured in vivo, followed by vortexing by inducing the
  • female porcines can be injected with PG600 and mature oocytes collected, typically,
  • the oocytes are preferably then enucleated.
  • this is preferably then enucleated.
  • metaphase II oocytes as determined by the presence of polar bodies, are preferably used
  • Enucleation may be effected before or after introduction of donor
  • Enucleation may be effected by known methods, such as
  • oocytes will be exposed to NCSU 23 medium (containing .2567
  • cytochalasin B After enucleation, the oocytes are placed in a suitable medium, e.g.,
  • metaphase II oocytes can be
  • HECM HECM
  • cytochalasin B 7.5 micrograms per milliliter cytochalasin B
  • a suitable medium for example an embryo culture medium such as NCSU
  • Enucleation may be accomplished microsurgically using a micropipette to remove
  • the oocytes are screened to identify those of
  • This screening is preferably effected by
  • a suitable dye e.g., 1 microgram per milliliter 33342 Hoechst
  • NCSU 23 can then be placed in a suitable culture medium, e.g., NCSU 23 and sucrose (.2567 mg/10
  • the recipient oocytes will preferably be enucleated at a
  • a single porcine differentiated cell e.g., a somatic or germ cell, pig cell or nucleus
  • the pig cell and the enucleated oocyte will be used
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to produce NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • the cells may be produced by NT units according to methods known in the art.
  • Electrofusion is accomplished by providing a pulse of
  • thermodynamic instability of such a small opening it enlarges until the two cells become
  • electrofusion media can be used including e.g., sucrose, mannitol, sorbitol and phosphate
  • Fusion can also be accomplished using Sendai virus as a fusogenic
  • the NT units prior to introduction into the fusion chamber, can be
  • the pig cell and oocyte can be electrofused by
  • a preferred protocol is to transfer optionally enucleated oocytes which
  • pronase preferably about 400 ⁇ l/1 well, and then diluted in
  • suitable media e.g., HECM/HEPES, and then centrifuged, preferably at about 6 kRPM
  • HECM/HEPES HECM/HEPES
  • the oocytes can be treated with TE using the same dissociation conditions
  • a suitable medium e.g., HECM/HEPES + sucrose (.2567 mg/10 ml).
  • NT embryos are then transferred to a suitable medium, e.g., NCSU
  • the NT embryos are then fused, preferably by applying 110V current for about thirty ⁇ seconds in a suitable fusion media.
  • 110V current for about thirty ⁇ seconds in a suitable fusion media.
  • the current preferred fusion medium comprises 500 ml of Sigma water, .28 mannitol
  • NCSU 23 NCSU 23
  • cytochalasin B (3 ⁇ l/2 ml) for about two hours prior to activation.
  • one or more caspase inhibitors may be used during maturation,
  • the NT unit may be activated by known methods. Activation may be effected
  • Such methods include, e.g., culturing the NT unit at sub-
  • shock to the NT unit This may be most conveniently done by culturing the NT unit at
  • Suitable activation protocols include the following:
  • Place oocytes or NT units typically about 22 to 28 hours post maturation in about
  • cytochalasin B preferably about eight hours, in 5 ⁇ g/ml of cytochalasin B and 20 ⁇ g/ml cycloheximide.
  • a current preferred method for effecting activation is in HECM/HEPES (H/H)
  • the NT fusions are placed in said H H BSA medium also containing 10 ⁇ m ionomycin
  • the NT fusions are again placed in H/H medium containing
  • the NT fusions are again placed in H/H containing
  • the activated NT units are then preferably
  • NCSU 23 medium e.g., NCSU 23 medium
  • FBS is preferably added and the NT units cultured to enable blastocyst formation.
  • the NT embryos can be transferred essentially immediately
  • the pig NT units can be activated in a 500 ⁇ m chamber by
  • NCSU 23 maintained in NCSU 23 with CB at 39°C and 5% C0 2 .
  • activation may be achieved by application of known activation
  • activation factor contained in sperm cells can activated NT units. Also, treatments such as
  • chemical activation can be effected about one to two hours after
  • the NT units are preferably
  • the NT units can be cultured for 3 to 4 hours
  • NCSU 23 plus CB in NCSU 23 plus CB, and thereafter in NCSU 23 without CB.
  • the NT units can be
  • the activated NT units may then be cultured in a suitable in vitro
  • TCM-199 + 10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP),
  • TCM-199 most common media used for the collection and maturation of oocytes.
  • serum supplement including fetal calf serum, newborn serum, estrual cow
  • a preferred maintenance medium includes TCM-199
  • the medium used is NCSU 23, and 2 to 5 days after activation
  • the NT units are cultured in fresh NCSU 23 and 5 to 10% fetal calf serum. Any of the
  • oviduct cells oviduct cells, BRL cells, uterine cells, and STO cells.
  • CR1 contains the nutritional substances necessary to support an embryo.
  • the NT units can be cultured in NCSU 23 plus 5 to 10% FCS until the NT units
  • NT units can be cultured until
  • NT embryos which are 1 cell can be introduced into
  • inventions can be effected using standard procedures used in the embryo transfer industry.
  • Synchronous transfers are desirable, i.e., the stage of the NT embryo is in synchrony with
  • helper embryos include normal porcine embryos,
  • parthenogenetic embryos activated, non-
  • helper embryos can vary significantly, i.e., from about one
  • helper embryo or tetraploid helper embryo is that the only offspring which will develop
  • DNA e.g., by use of radiolabeled antibody or other probe.
  • the present invention is particularly useful for producing cloned
  • the present invention is advantageous in that
  • transgenic procedures can be simplified by working with a differentiated cell source that
  • the differentiated cells used for donor nuclei can be clonally propagated.
  • the differentiated cells used for donor nuclei can be clonally propagated.
  • differentiated cells are then used for nuclear transplantation with enucleated oocytes.
  • a mammalian cell from a mammalian cell may be used for altering the differentiated cell to be used as the
  • DNA sequence may be heterologous. Included is the technique of homologous
  • porcine genes will be "knocked out” and the human homolog, e.g., a DNA sequence
  • an immunological protein encoding an immunological protein, hormone, structural protein, clotting factor, enzyme,
  • the present invention can thus be used to provide adult pigs with desired
  • traits is particularly useful, including transgenic or genetically engineered animals, and
  • the present invention will allow production of single sex offspring, and production of pigs having improved meat production, reproductive traits
  • cell and tissues from the NT fetus including
  • transgenic and/or chimeric fetuses can be used in cell, tissue and organ transplantation
  • transgenic pigs have uses including models for diseases,
  • endogenous structural genes e.g.,
  • porcine serum albumin gene will be replaced by HSA gene.
  • the cells are mechanically removed from the zone and are then used. This is
  • a feeder layer e.g., uradiated fibroblast cells.
  • a feeder layer e.g., uradiated fibroblast cells.
  • the cells are maintained in the feeder layer in a suitable growth
  • alpha MEM e.g., alpha MEM supplemented with 10% FCS and 0.1 mM ⁇ -mercaptoethanol
  • CICM cells may be genetically engineered or transgenic pig CICM cells.
  • genetically engineered or transgenic pig CICM cells may be used.
  • the resultant CICM cells and cell lines have numerous therapeutic and diagnostic features
  • CICM cells may be used for cell transplantation
  • mouse embryonic stem (ES) cells are capable of ES cells
  • CICM cells produced according to the invention should possess similar differentiation
  • the CICM cells according to the invention will be induced to differentiate to
  • the subject pig obtains the desired cell types according to known methods.
  • the subject pig obtains the desired cell types according to known methods.
  • the subject pig obtains the desired cell types according to known methods.
  • CICM cells may be induced to differentiate into hematopoietic stem cells, neural cells,
  • muscle cells cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neural cells, etc., by culturing such cells in differentiation medium and under
  • stem cells subjecting stem cells to an induction procedure comprising initially culturing aggregates
  • references are exemplary of reported methods for obtaining differentiated cells from
  • the subject CICM cells including genetically engineered or transgenic CICM cells, to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic
  • the subject CICM cells may be used to obtain any desired differentiated cell type.
  • hematopoietic stem cells may be used in medical treatments requiring bone manow
  • Hematopoietic stem cells can be obtained, e.g., by fusing adult
  • somatic cells of a cancer or AIDS patient e.g., epithelial cells or lymphocytes with an
  • Such hematopoietic cells may be used in the treatment of diseases including cancer and
  • the present invention can be used to replace defective genes, e.g., defective
  • therapeutically beneficial proteins such as growth factors, lymphokines, cytokines,
  • DNA sequences which may be introduced into the subject CICM cells include, by
  • neurotrophin-3 neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokines
  • the present invention includes the use of pig cells in the treatment of human
  • pig CICM cells, NT fetuses and NT and chimeric offspring transgenic
  • CICM cell fetuses and offspring
  • brain cells from pig For example, brain cells from pig
  • NT fetuses may be used to treat Parkinson's disease.
  • the subject CICM cells may be used as an in vitro model of differentiation
  • subject CICM cells may be used as nuclear donors for the production
  • NCSU 37 Modified NCSU 37 Medium
  • G/Strept is optional.
  • EGF Stock Epidermal Growth Factor from 10 ng/ ⁇ l EGF stock
  • Follicles are graded visually for size. Follicles that are 3 mm x 3 mm up to 7 mm
  • a 10 cc syringe with an 18 gauge needle is preferably used to draw up 1 ml of
  • the needle is then positioned bevel down and pushed into the follicle with slight
  • the needle is then removed from the syringe and the follicular fluid is
  • the tube in order to avoid stripping of the cumulus cells as well as damaging the oocytes.
  • Porcine follicular fluid typically about 3-6 mm follicles
  • the oocytes and follicular cells are allowed to settle therefrom, e.g., by
  • Equine Chorionic Gonadotropin and human Chorionic Gonadotropin Stock for MAT (TMSG/hCG ⁇ and Preparation
  • This material is diluted from 6000 IU to 2000 IU/ml by the addition of 3 ml dH 2 0.
  • hCG Chaorulon; Intervet Inc.
  • the hCG material is diluted from 10,000 IU to 2000 IU/ml by the addition of 5 ml
  • Follicular fluid pFF is aspirated and saved for use in culture system if needed
  • the oviduct is dissected from the uterus and flushed with buffer media.
  • eggs are loaded into a 5 X A inch Tom Cat catheter (Sovereign Cat.
  • each oviduct typically no more than 100 NTs are transfened per animal.
  • Caspase inhibitors optionally may be included in the buffer
  • the gilts are anesthetized using
  • Pregnancy check is preferably performed thirty days after surgery, typically by
  • the fetuses can be retrieved at that time by C-section, and analyzed by PCR
  • OCCs are resuspended in 20 ml Hepes-PVA and allowed to settle; repeat 2 times.
  • OCCs are moved to grid dishes and selected for culture. Selected OCCs
  • the washed OCCs (about 50) are placed in a four-well Nunc plate tje we;;s pf
  • the oocytes are then placed in the same medium except lacking hormones for
  • porcine fibroblasts Primary cultures of porcine fibroblasts are obtained from pig fetuses 30 to 114
  • trypsin EDTA solution 0.05% trypsin/0.02% EDTA; GIBCO, Grand Island,
  • Fibroblast cells are plated in tissue culture dishes and cultured in fibroblast growth medium (FGM) containing: alpha-MEM medium (BioWhittaker, Walkersville, MD)
  • FCS fetal calf serum
  • the fibroblasts are grown and maintained in a
  • tissue is incubated overnight at 10 °C in trypsin EDTA solution (0.05% trypsin/0.02%
  • alpha-MEM medium BioWhittaker, Walkersville, MD
  • FCS fetal calf serum
  • the fibroblast cells can be isolated at virtually any time.
  • fetal fibroblasts which may be used as donor nuclei are:
  • fibroblast cells into the enucleated pig oocyte.
  • fibroblast cells are synchronized in Gl or GO of the
  • the fibroblast cells are grown to confluence. Then the concentration of fetal
  • donor cells e.g., fibroblasts can be obtained directly from a live
  • animal e.g., an adult porcine, e.g., from a tissue or fluid source.
  • the oocytes can preferably be enucleated. Removal of cumulus cells is
  • oocytes can be removed and placed in HECM (Seshagiri and
  • the stripped oocytes are then screened for
  • a cunent prefened procedure comprises exposure of oocytes to NCSU 23 +
  • sucrose + HXT for at least 20 minutes, followed by enucleation effected in H/H medium
  • the oocytes are preferably
  • NCSU 23 containing sucrose before transfer.
  • Cells for transfer are preferably treated with pronase or TE.
  • pronase In the case of pronase,
  • the cells are treated with 400 ⁇ l/1 well, allowed to incubate and then diluted in H/H and
  • Transfer is then preferably
  • sucrose for fusion
  • Nuclear transfer units are preferably put through a gradient of H/H and mannitol
  • the cells are electrofused, by placing cells for 30 ⁇ sec @100 V. After fusion, the
  • NT units are placed in pure H/H and then into NCSU 23 + Cyto B (3 ⁇ l/2ml), preferably
  • activation may be effected prior, proximate, or after
  • NT units are placed in H/H medium containing 1 mg/ml BSA + 10 ⁇ M ionomycin
  • the NT units are placed in the same H/H medium
  • the NT units are placed in the same H/H medium containing 1 mg/ml
  • the NT units are then rinsed to remove DMAP, and then cultured in NCSU 23.
  • the media is changed on day three and 5% FBS is added late on day five, and the NT
  • NT units cultured until blastocysts form. 2. Single activation pulse. NT units are removed from the NCSU 23 plus CB and

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Abstract

L'invention concerne un procédé amélioré de transfert nucléaire impliquant la transplantation de noyaux de cellules de porcs différenciées de donneurs dans des ovocytes de porcs énucléés. Les unités de transfert nucléaire qui en résultent servent à la multiplication de génotypes et de génotypes transgéniques par production de foetus et de progéniture. Ce procédé facilite la production d'embryons, de foetus et de progéniture de porcs génétiquement modifiés ou transgéniques, puisque la source de cellules différenciées du noyau donneur peut être génétiquement modifiée et propagée par clonage.
PCT/US2000/024958 1999-09-13 2000-09-13 Clonage de porcs a l'aide de cellules ou de noyaux de donneurs provenant de cellules differenciees et production de porcins multipotents WO2001019181A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
BR0013959-9A BR0013959A (pt) 1999-09-13 2000-09-13 Clonagem de porcos usando células ou núcleos doadores a partir de células diferenciadas e produção de porcino pluripotente
JP2001522835A JP2003509031A (ja) 1999-09-13 2000-09-13 ドナー細胞又は分化細胞からの核を使用する豚のクローニング並びに多能性豚の産生
EP00961827A EP1241931A4 (fr) 1999-09-13 2000-09-13 Clonage de porcs a l'aide de cellules ou de noyaux de donneurs provenant de cellules differenciees et production de porcins multipotents
CA002381124A CA2381124A1 (fr) 1999-09-13 2000-09-13 Clonage de porcs a l'aide de cellules ou de noyaux de donneurs provenant de cellules differenciees et production de porcins multipotents
IL14850800A IL148508A0 (en) 1999-09-13 2000-09-13 Cloning pigs using donor cells of nuclei from differentiated cells and production of pluripotent porcine
AU73725/00A AU7372500A (en) 1999-09-13 2000-09-13 Cloning pigs using donor cells or nuclei from differentiated cells and production of pluripotent porcine.
MXPA02002653A MXPA02002653A (es) 1999-09-13 2000-09-13 Clonacion de cerdos usando celulas donadoras o nucleos a partir de celulas diferenciadas y produccion de porcinos pluripotentes.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/394,902 1999-09-13
US09/394,902 US7291764B1 (en) 1997-01-10 1999-09-13 Cloning pigs using non-quiescent differentiated donor cells or nuclei

Publications (1)

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WO2001019181A1 true WO2001019181A1 (fr) 2001-03-22

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Country Status (9)

Country Link
EP (1) EP1241931A4 (fr)
JP (1) JP2003509031A (fr)
CN (1) CN1377225A (fr)
AU (1) AU7372500A (fr)
BR (1) BR0013959A (fr)
CA (1) CA2381124A1 (fr)
IL (1) IL148508A0 (fr)
MX (1) MXPA02002653A (fr)
WO (1) WO2001019181A1 (fr)

Cited By (5)

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US20130150465A1 (en) * 2007-03-07 2013-06-13 Aarhus Universitet Pig model for breast cancer, mitochondria related protein folding disorders and/or epidermolysis bullosa simplex
WO2016094679A1 (fr) * 2014-12-10 2016-06-16 Regents Of The University Of Minnesota Cellules, tissus et organes génétiquement modifiés pour le traitement d'une maladie
WO2017218714A1 (fr) * 2016-06-14 2017-12-21 Regents Of The University Of Minnesota Cellules, tissus et organes génétiquement modifiés pour le traitement d'une maladie
US11246299B2 (en) 2015-03-04 2022-02-15 Pormedtec Co., Ltd. Disease model pig exhibiting stable phenotype, and production method thereof
CN117044679A (zh) * 2023-10-13 2023-11-14 成都铁骑力士饲料有限公司 一种血清fas在川藏黑猪s05系瘦肉型猪选育中的应用

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JP2004275074A (ja) * 2003-03-14 2004-10-07 Nipro Corp 動物胚における正常性のスクリーニング方法
CN100404675C (zh) * 2006-02-22 2008-07-23 李宁 一种生产体细胞克隆猪的方法
CN111349615B (zh) * 2018-12-24 2024-08-13 上海细胞治疗集团股份有限公司 制备过表达外源基因的细胞的方法
CN115305260B (zh) * 2021-11-30 2023-09-22 海南大学 一种金鲳鱼受精卵的显微注射方法及应用
CN116640804B (zh) * 2023-04-20 2024-11-19 云南农业大学 一种基于体细胞核移植技术构建三倍体猪的方法

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WO1997037009A1 (fr) * 1996-04-01 1997-10-09 University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, Represented By Its Amherst Campus Lignees de cellules a bouton embryonnaire cultive, derivees d'embryons d'ongules
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130150465A1 (en) * 2007-03-07 2013-06-13 Aarhus Universitet Pig model for breast cancer, mitochondria related protein folding disorders and/or epidermolysis bullosa simplex
KR102656470B1 (ko) 2014-12-10 2024-04-09 리전츠 오브 더 유니버스티 오브 미네소타 질환을 치료하기 위한 유전적으로 변형된 세포, 조직 및 장기
WO2016094679A1 (fr) * 2014-12-10 2016-06-16 Regents Of The University Of Minnesota Cellules, tissus et organes génétiquement modifiés pour le traitement d'une maladie
KR20170092692A (ko) * 2014-12-10 2017-08-11 리전츠 오브 더 유니버스티 오브 미네소타 질환을 치료하기 위한 유전적으로 변형된 세포, 조직 및 장기
US9888673B2 (en) 2014-12-10 2018-02-13 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10278372B2 (en) 2014-12-10 2019-05-07 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US10993419B2 (en) 2014-12-10 2021-05-04 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US11234418B2 (en) 2014-12-10 2022-02-01 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
US11246299B2 (en) 2015-03-04 2022-02-15 Pormedtec Co., Ltd. Disease model pig exhibiting stable phenotype, and production method thereof
WO2017218714A1 (fr) * 2016-06-14 2017-12-21 Regents Of The University Of Minnesota Cellules, tissus et organes génétiquement modifiés pour le traitement d'une maladie
AU2017285224B2 (en) * 2016-06-14 2023-05-18 Regents Of The University Of Minnesota Genetically modified cells, tissues, and organs for treating disease
CN117044679A (zh) * 2023-10-13 2023-11-14 成都铁骑力士饲料有限公司 一种血清fas在川藏黑猪s05系瘦肉型猪选育中的应用
CN117044679B (zh) * 2023-10-13 2024-01-19 成都铁骑力士饲料有限公司 一种血清fas在川藏黑猪s05系瘦肉型猪选育中的应用

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IL148508A0 (en) 2002-09-12
CA2381124A1 (fr) 2000-09-13
AU7372500A (en) 2001-04-17
JP2003509031A (ja) 2003-03-11
EP1241931A4 (fr) 2004-12-29
MXPA02002653A (es) 2003-10-14
BR0013959A (pt) 2002-05-21
EP1241931A1 (fr) 2002-09-25
CN1377225A (zh) 2002-10-30

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