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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