CN1299408A - Embryonic or stem-like cell links produced by cross-species nuclear transplantation - Google Patents
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Abstract
An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.
Description
Invention field
The present invention relates generally to produce protoblast or stem cell-like cell by the enucleation oocyte of the nucleus of animal or human's cell being implanted with donor nuclei animal not of the same race.The present invention relates more specifically to by the animal ovocyte with the renucleation stoning of primate or people's cell, as the ovocyte of primate or tool pawl animal, produce primate or person embryo cell or stem cell-like cell for the ox enucleation oocyte in preferred embodiments.
The invention still further relates to the protoblast or the stem cell-like cell of gained, the protoblast or the stem cell-like cell that are preferably primate or people are used for the treatment of, diagnosis, generation can be used for the noble cells for the treatment of or diagnosing, with genetically modified embryo of generation or genetically modified noble cells, clone, the purposes of tissue and organ.In addition, the protoblast of gained of the present invention or stem cell-like cell itself also can be used to produce mosaic or clone, is preferably in the nuclear transplantation of genetically modified clone or chimaeric animals or the consideration convey shifting method and is used as nuclear donor.
Background of invention
External, obtaining method that the embryo does (ES) clone by the mouse body early embryo before implanting is that well-known (example is seen Evans etc., nature, 29:154-156 (1981); Martin, Proc.Natl. Acad.Sci., USA, 78:7634-7638 (1981)), if having inoblast feeder layer (Evans etc., document is the same) or differentiation inhibition source (Smith etc., developmental biology, 121:1-9 (1987)), the ES cell can undifferentiated state be gone down to posterity.
It is reported that the ES cell serves many purposes, for example, the ES cell can be used as the vitro differentiation model, especially can be used as the gene in vitro model that research participates in the early development adjusting.When preceding mice embryonic is implanted in the mouse ES cells importing, can produce germline mosaic, thereby illustrate its versatility (Bradley etc., nature, 309:255-256 (1984)).
Because the ES cell can be transferred to the next generation with its genome, therefore, the ES cell has the potential practicality, and it kind is operation that the ES cell that has or do not have a required genetic modification by use can carry out domestic animal.In addition, to domestic animal, as tool pawl animal, from support growth on schedule (Smith etc., biological self reproducing, the 40:1027-1035 (1989) of enucleation oocyte such as the nuclear energy that obtains the livestock embryo before implanting; Keefer etc., biological self reproducing, 50:935-939 (1994)).This is with to derive from the nuclear phase of mice embryonic anti-, it is reported that the nuclear that derives from mice embryonic still can not support the growth (Cheong etc., biological self reproducing, 48:958 (1993)) of enucleation oocyte behind the 8-cell stage after the transfer.Therefore, the ES cell that derives from domestic animal caters to the need very much because they can provide potential through genetic manipulation or otherwise can be used for examining the totipotency donor nuclei source of transfer method.
Some research groups have reported the separation of the embryonic lineage that is stated to be versatility.For example, according to Notarianni etc., J.Reprod.Fert.Suppl, 43:255-260 (1991) report, by pig and sheep blastocyst set up be stated to be stable, some morphology that the clone of versatility, wherein said blastocyst show and growth characteristics are similar to by the cell of immunity excision by the isolated inner cell mass primary culture of sheep blastocyst.In addition, Notarianni etc., J.Reprod.Fert.Suppl, 41:51-56 (1990) disclose keeping of the versatility embryonic lineage of inferring that derives from the pig blastocyst and have broken up.In addition, Gerfen etc., Animal Biotechnology, 6 (1): 1-14 (1995) discloses by the pig blastocyst and has separated embryonic lineage.Need not the working conditions substratum, these cells can be on the mouse embryo fibroblasts feeder layer stable maintenance.It is reported that these cells are divided into several different cell types (Gerfen etc., document is the same) in the process of cultivating.
In addition, Saito etc., Roux ' s Arch.Dev.Biol., 201:134-141 (1992) have reported the ox embryonic stem cell-like cell system through cultivating, and this clone can be survived for 3 generations, but promptly disappeared after the 4th generation.Handyside etc., Roux ' s Arch.Dev.Biol., 196:185-190 (1987) disclose to cultivate under the condition that allows to separate the mouse ES cells system that derives from mouse ICM and have excised isolating sheep embryo inner cell mass through immunity.According to Handyside etc., (1987) (document is the same) report, under these conditions, sheep ICM adheres to, diffusion and produce ES cell sample and entoderm like cell zone, but through prolong cultivate after, have only the entoderm like cell high-visible.
Recently, Cherny etc., Theriogenology, 41:175 (1994) reported can in extended culture, keep be stated to be versatility derived from the genitaloid clone of ox.After cultivating about 7 days, these cells have produced ES sample colony, and the dyeing of its alkaline phosphatase (AP) is positive, and described cell also shows the ability that forms embryoid and be divided at least two kinds of different cell types automatically.It is reported that these cells have also been expressed transcription factor OCT4, the mRNA of OCT6 and HES1, and it is believed that to have only the ES cell can express this homoeobox gene pattern.
Recently, according to Campbell etc., nature, 380:64-68 (1996) report, blastodisc (ED) cell through cultivating is carried out consideration convey produced the lamb that lives after moving, wherein said ED cell gets comfortable impelling and separates the sheep embryo of cultivating under the condition that mouse ES cells is in 9 day age, and the author draws following results according to experimental result: promptly move by consideration convey, the ED cell that derives from sheep embryo in 9 day age is totipotent, and should all can performance keep in cultivation.
Van Stekelenburg-Hamers etc., Mol.Reprod.Dev., 40:444-454 (1995) has reported separation that is stated to be permanent cell line and the evaluation that derives from ox blastocyst inner cell mass cell.The author separates under different conditions and has cultivated the ICM that derives from ox blastocyst in 8 or 9 day age and can support adhering to and hypertrophy of ox ICM cell most effectively to determine which kind of feeder cell and substratum.They reach a conclusion according to experimental result: use STO (l cell) feeder cell (to substitute the cattle uterus epithelial cell) and use the serum (rather than normal serum) of charcoal-peel off to be added into can strengthen in the substratum adhering to and hypertrophy through the ICM cell of cultivation.Yet according to Van Stekelenburg etc., above-mentioned clone more is similar to the ICM cell of epithelial cell rather than versatility.
In addition, (WO94/24274 such as Smith, on October 27th, 1994 is open), (WO90/03432 such as Evans, April 5 nineteen ninety is open) and (WO94/26889 such as Wheeler, on November 24th, 1994 is open) reported the separation of stem cell animal, select and propagation, it is said that this stem cell can be used for obtaining transgenic animal.Evans etc. (WO90/03432, April 5 nineteen ninety open) have reported also that the embryonic stem cell that is obtained being stated to be versatility by pig and Niu Yansheng, this embryonic stem cell it is said and can be used for producing transgenic animal.In addition, Wheeler etc. (WO94/26884, on November 24th, 1994 open) disclose and it is said the embryonic stem cell that can be used for preparing chimeric and genetically modified tool pawl animal.Therefore, according to above-mentioned, a lot of research groups obviously want to attempt producing ES clone, because ES clone can be used to produce the clone's or genetically modified embryo and can be used for nuclear transplantation potentially.
Also the someone has reported and has made the ICM cell of apparatus pawl animal carry out nuclear transplantation.For example, Collas etc., Mol.Reprod.Dev., 38:264-267 (1994) disclose by will be through the micro-injection of cracked donorcells to the mature oocyte of stoning and ox ICM is carried out nuclear transplantation.The document disclose external with embryo culture 7 days to produce 15 blastocysts, by being transferred to Niu Shouti, cause 4 gestation and 2 births.Keefer etc., biological self reproducing, 50:935-939 (1994) disclose in the consideration convey shifting method, use ox ICM cell as donor nuclei to produce blastocyst, by migrating to Niu Shouti, cause producing the offspring of several work.In addition, Sims etc., Proc.Natl.Acad.Sci., USA, 90:6143-6147 (1993) disclose by producing calf in the mature oocyte that will move to stoning through the consideration convey of the ox ICM of short-term vitro culture cell.
In addition, also the someone has reported that the blastoderm cells through cultivating is carried out consideration convey has produced the lamb that lives (Campbell etc., naturally, 380:64-68 (1996)) after moving.Somebody's report: in consideration convey moves, use ox versatility protoblast and produce chimeric fetus (Stice etc., biological self reproducing, 54:100-110 (1996); Collas etc., Mol.Reprod.Dev., 38:264-267 (1994)).
In addition, also the someone attempts producing xenogeneic (cross species) NT unit (Wolfe etc., Theriogenology, 33:350 (1990)).Specifically, be that the ovocyte of ox protoblast and wild ox is merged to produce the xenogeneic NT unit that some may have inner cell mass.Yet the donor nuclei that uses in the consideration convey shifting method is derived from protoblast rather than adult cell.In theory, protoblast is easier to reprogramming than adult cell.The research belongs to same period (referring to Diberardino, differentiation, 17:17-30 (1980)) with the NT research of carrying out in early days in the frog.The research is included in very similar animal (ovocyte of ox nucleus and wild ox) in the phylogeny.In contrast, when fusion has more multifarious kind in the NT process (the ox nuclear transplantation is to the hamster ovocyte), do not obtain the inner cell mass structure.In addition, nobody reports that the inner cell mass that derives from NT unit can be used to form the ES cell sample colony that can be bred in the past.
Collas etc. (document is the same) have instructed use granulosa cell (adult body cell) to produce the ox consideration convey and have moved the embryo.Yet different with the present invention is, these experiments do not relate to xenogeneic consideration convey and move, and different with the present invention be not obtain ES like cell colony.
Recently, United States Patent (USP) 5,843,780 (were issued to James A.Thomson on December 1st, 1998, transfer the possession of WARF subsequently in Wisconsin Alumni) claim the primate embryonic stem cells goods that disclose purifying, it can (ⅰ) breed more than 1 year in vitro culture; (ⅱ) keep caryogram, wherein all karyomit(e) characteristics of primate all exist and not significant variation the in prolonging the process of cultivating; (ⅲ) in culturing process, keep being divided into entoderm, the potentiality of mesoderm and ectoderm tissue derived thing; (ⅳ) when on the inoblast feeder layer, cultivating, can not break up.Qian report according to this, the SSEA-1 of these cells is labeled as feminine gender, SEA-3 is labeled as the positive, SSEA-4 is labeled as the positive, and these cells can be expressed alkaline phosphatase activities now, be versatility, and have the existence of all karyomit(e) characteristics that comprise primate and the caryogram that changes to some extent of none karyomit(e) wherein.In addition, the TRA-1-60 of these cells and TRA-1-81 are labeled as the positive.It is said that when being injected to the SCID mouse, this cell can be divided into entoderm, mesoderm and ectoderm cell.Thomson etc. are in science, and 282:1145-1147 and Proc.Natl.Acad.Sci. have also discussed it is said derived from human or the paotoblastic embryonic stem cell line of primate among the USA 92:7844-7848 (1995).
Therefore, Thomson discloses protoblast or stem cell-like cell and the production method thereof that is stated to be non-human primate and people.Yet, still need to study the person embryo cell of transplant recipient self or the production method of stem cell-like cell at present, because described cell has significant treatment and diagnosis potentiality.
In this regard, identified multiple can be by the human diseases of cellular transplantation therapy.For example, Parkinson's disease is to be degenerated by the dopaminergic neuron in the black substance to cause.Parkinsonian standard treatment comprises uses L-DOPA, the loss that this can the respite Dopamine HCL, but can cause severe side effect, finally can not reverse the process of disease.The parkinsonian different methods of another kind of treatment it is said and having been widely used aspect a lot of encephalopathics of treatment and the central nervous system injury that this method comprises to be implanted the cell or tissue of fetus or new born animal in the brain of growing up.The neurone that derives from a plurality of brains of fetus district can mix in the brain of growing up.This graft can be alleviated laboratory animal through experiment inductive behavioral deficiency, comprises complicated cognitive function defective.The initial test-results that is obtained by people's clinical trial also is likely.Yet, very limited by the human fetal cell or tissue supply that miscarriage obtains, in addition, obtain cell or tissue by aborted fetus and caused that people argue widely.
At present, also do not produce the method for " fetus-sample " cell by the patient.In addition, allograft tissue also is not easy to obtain, and allotransplantation and heteroplastic organizing all will suffer transplant rejection.In addition, in some cases, to carry out genetic modification be favourable for pair cell or tissue before transplanting.Yet much the cell or tissue through essential genetic modification can not divide in culturing process preferably, and most gene transformation type needs quick splitted cell.
Therefore, obviously need the cell of supply person embryo cell or stem-like cell undifferentiated to be used for transplanting and cell and gene therapy in this area.
Goal of the invention
The purpose of this invention is to provide new and the generation protoblast of improvement or the method for stem cell-like cell.
The present invention's purpose more specifically provides the new generation protoblast or the method for stem cell-like cell, and described method comprises in the renucleation xenogenesis enucleation oocyte with Mammals or people's cell.
Of the present invention another more specifically purpose provide the new generation non-human primate or the method for person embryo cell or stem cell-like cell, described method comprises in animal or human's ovocyte with the renucleation stoning of non-human primate or people's cell, as tool pawl animal, in people or the primate non-nucleus egg mother cell.
Of the present invention another more specifically purpose provide the new generation pedigree-damaged non-human primate or the method for person embryo cell or stem cell-like cell, described method comprises non-human primate or people's cell, as in the non-human primate or human oocyte of the renucleation stoning of the cell of being grown up, wherein this cell can not be divided into specific cell lineage through genetic modification, perhaps modify and make cell become " dead ", thereby can not produce the offspring of survival by for example antisence DNA or ribozyme telomerase gene.
Another object of the present invention is to strengthen the effectiveness that consideration convey moves, especially undertaken genetic engineering modified by consideration convey being moved used donor somatocyte to provide the gene that strengthens fetal development (as the gene of MHC I family, especially Ped gene is as Q7 and/or Q9) thus expression strengthen the growth that moves the pre-implantation embryos of generation by consideration convey.
Another object of the present invention is to strengthen the generation that consideration convey moves the embryo by IVP, more specifically for carrying out genetic modification and make cell pair cell apoptosis have resistance to strengthen the generation that consideration convey moves the embryo by consideration convey being moved used donorcells, for example by import can provide DNA construct that the gene (as Bcl-2 or Bcl-2 family member) that suppresses apoptosis expresses and/or by expression be specific to can be in the early embryo development process antisense ribozyme of the gene of cell death inducing to strengthen the generation that consideration convey moves the embryo.
But another object of the present invention is to make their expression codings and mark remember the DNA construct of the specific cells cyclin that (as macroscopic fluorescent mark albumen) links to each other by donorcells being carried out genetic modification, thereby improve the specific cells cycle, finally improve the effectiveness that consideration convey moves as the selection of the donorcells of G1 phase.
Another object of the present invention is by at one or more proteinase inhibitor, is preferably the following embryo who cultivates external generation of existence of one or more capsase inhibitor, thereby suppresses apoptosis to strengthen described embryo's growth.
Another object of the present invention provides protoblast or the stem cell-like cell that produces by in the renucleation xenogenesis enucleation oocyte with animal or human's cell.
Of the present invention another more specifically purpose provide by animal ovocyte the renucleation stoning of primate or people's cell, as the people, in the enucleation oocyte of primate or tool pawl animal and the primate or person embryo cell or the stem cell-like cell that produce.
Another object of the present invention is described protoblast or stem cell-like cell are used for the treatment of or diagnose.
Specific purposes of the present invention are to use described primate or person embryo cell or stem cell-like cell treatment or diagnose any cell that carries out, and tissue or organ transplantation are to its treatment or diagnose favourable disease.
Protoblast or stem cell-like cell that another concrete purpose of the present invention is to use the present invention to produce produce the cell of differentiation, tissue or organ.
The present invention's purpose more specifically is to use primate that the present invention produces or person embryo cell or stem cell-like cell to produce people's cell of differentiation, tissue or organ.
Protoblast or stem cell-like cell that another concrete purpose of the present invention is to use the present invention to produce produce through genetic engineering modified protoblast or stem cell-like cell, this cell can be used for producing through people's cell genetic engineering modified or genetically modified differentiation, tissue or organ for example are used for gene therapy.
Another concrete purpose of the present invention is that the protoblast of the external generation of the present invention or stem cell-like cell are used for for example studying cytodifferentiation and detection applications (as being used for drug research).
Another object of the present invention provides the transplantation therapy of improvement, and described method comprises that protoblast that use is produced by the present invention or stem cell-like cell produce waits gene or homogenic cell, organizes or organ.The treatable disease of described therapy for example comprises: Parkinson's disease, Huntington, presenile dementia, ALS, Spinal injury, multiple sclerosis, muscular dystrophy, diabetes, hepatopathy, heart trouble, cartilage is replaced, burn, vascular disease, urinary tract disease, and immunodeficiency, bone marrow transplantation, diseases such as cancer.
Another object of the present invention is to be used for gene therapy with produce genetically modified of the present invention or through the protoblast or the stem cell-like cell of genetic modification, in particular for treating and/or preventing above-mentioned disease and damage.
Another object of the present invention be produce genetically modified of protoblast that the present invention is produced or stem cell-like cell or the present invention or through the protoblast of genetic modification or stem cell-like cell as used nuclear donor in the nuclear transplantation.
What another object of the present invention was to use that the present invention produces produces transgenic animal through the ES of genetic modification cell, as non-human primate, and rodent, tool pawl animal etc.Described transgenic animal can be used for producing for example to be studied human diseases or produces required polypeptide, as therapeutical agent or the required animal model of nutrient drug.
Above and other objects of the present invention, advantage and feature be particular embodiment hereinafter, therefore, will more be expressly understood feature of the present invention with reference to following detailed description and appended claims about the preferred embodiment of the invention.
The accompanying drawing summary
Fig. 1 is the photo that moves (NT) unit by the consideration convey that the cell transfer of will being grown up produces to the bovine oocyte of stoning.
Fig. 2 to 5 is the photos that derive from the embryonic stem cell like cell of NT unit shown in Figure 1.
Detailed Description Of The Invention
The invention provides by consideration convey and move or nuclear transfer generation blastocyte or stem cell-like cell, more specifically is the new method of non-human primate or people's blastocyte or stem cell-like cell. In this application, consideration convey moves or nuclear transfer or NT can Alternates.
As mentioned above, move by consideration convey or nuclear transfer separates real blastocyte or stem cell-like cell there is no people's report. On the contrary, the ES-like cell of report separates from the embryo who is fertilized in the past. In addition, never the someone reported cell or the DNA that comprises the dissimilar kind of gene, more specifically was that the consideration convey of success that comprises the egg mother cell of the adult cell of a kind (such as the people) or DNA and another uncorrelated kind moves. On the contrary, possessor's report has produced the embryo by the cell that merges closely related kind to the greatest extent, and such as ox-goat and ox-wild ox, but they can not produce ES cell (Wolfe etc., Theriogenology, 33 (1): 350 (1990)). In addition, also nobody reported the method that is produced primate or people's ES cell by non-fetal tissue source. On the contrary, at present available limited human fetal cell and tissue must derive from or derived from the tissue of spontaneous abortion and the fetus of miscarriage.
Before the present invention, nobody can obtain blastocyte or in the cell like cell by xenogeneic nuclear transfer.
The inventor finds very unexpectedly by in the animal egg mother cell with the renucleation stoning of people's cell (such as people's cell of the differentiation of growing up), move (NT) unit for generation of consideration convey, cell wherein can produce person embryo cell or stem cell-like cell and cell colony by cultivating, and can obtain person embryo cell or stem cell-like cell and cell colony. This result is very wondrous, because this result has illustrated nuclear transfer between effective xenogenesis first, it comprises the enucleation oocyte with dissimilar kind on the donorcells of differentiation or the nuclear quiding gene, for example with the animal or human's cell that breaks up, implant such as the nucleus of adult cell in the stoning ovum of different animals kind to produce nuclear transplantation unit, when cultivating wherein contained cell under proper condition, can produce blastocyte or stem cell-like cell and cell colony.
Preferably will be cultured to for generation of the NT unit of ES like cell size and be at least 2 to 400 cells, 4 to 128 cells more preferably, most preferably size is at least about 50 cells.
In the present invention, blastocyte or stem cell-like cell refer to the cell that the present invention produces. The reason that among the application these cells is called stem cell-like cell rather than stem cell is that they generally are to move generation by xenogeneic consideration convey. Although expect that these cells have the differentiation capability similar to normal stem cell, the particularity of the mode that produces owing to their makes them also have some inapparent differences. For example, these stem cell-like cells have the mitochondria that consideration convey moves used egg mother cell, and therefore, its behavior is different from conventional embryonic stem cell.
This discovery is based on following observed result: the cell of will being grown up, when the HE nucleus that especially derives from people's donor oral cavity is implanted the bovine oocyte of stoning, cause forming nuclear transplantation unit, can produce human stem cell like cell or blastocyte and person embryo cell or stem cell-like cell colony by the cell of cultivating wherein. Recently, implant in the bovine oocyte of stoning by becoming human keratinized cell, successfully produced blastocyst and ES clone and reproduced the above results. According to the above results, the inventor proposes following hypothesis, be bovine oocyte and human oocyte and may be that general mammal ovocyte must be through the process of post-mature in embryo development procedure, this maturation be enough similar or conservative so that bovine oocyte can be used as effective substitute of human oocyte. Obviously, egg mother cell in fact generally contains protein or the nucleic acid factor, and inducing embryo is grown under proper condition, and these functions in not of the same race are identical or very similar. These factors can contain material RNA and/or Telomerase.
Can effectively implant this fact of bovine oocyte according to people's nucleus, can reasonably expect implantable other the irrelevant kind of people's cell, in the egg mother cell such as other tool pawl animal and other animal. Especially, the egg mother cell of other tool pawl animal should be fit to, pig for example, sheep, horse, goat etc. In addition, the egg mother cell in other source should be fit to, and for example derives from other primate, amphibian animal, rodent, rabbit, the egg mother cell of cavy etc. In addition, use similar method, should be transferred to people's cell or nucleus in the human oocyte and use the blastocyte of gained to produce people ES cell.
Therefore, in the widest embodiment, the present invention includes by injection or merge animal or human's nucleus or animal or human's cell are implanted in the enucleation oocyte of the animal kind that is different from donor nuclei to produce NT unit, contained cell can be used for obtaining blastocyte or stem cell-like cell and/or cell culture in this NT unit. For example, the present invention includes by injection or merging implants the nucleus of tool pawl animal or the cell of tool pawl animal another kind of, in the enucleation oocyte such as another kind of tool pawl animal or non-tool pawl animal, cell and/or nuclear mix generation NT unit, the cell of cultivating under proper condition NT unit can obtain cellulous NT unit, preferred its contains at least about 2 to 400 cells, and more preferably 4 to 128 cells most preferably are at least about 50 cells. The cell of this NT unit can be used for producing blastocyte or stem cell-like cell or cell colony after cultivating.
Yet, the preferred embodiments of the invention comprise: by the people that nuclear or people's cell of donor people cell are implanted stoning, the egg mother cell of primate or non-human primate animal, such as the egg mother cell of tool pawl animal, in preferred embodiments for producing non-human primate or person embryo cell or stem cell-like cell in the ox non-nucleus egg mother cell.
Usually, can produce blastocyte or stem cell-like cell by the consideration convey shifting method that comprises the following steps:
(ⅰ) obtain required human or animal's cell with the source (can carry out genetic modification to it) as donor nuclei;
(ⅱ) from appropriate sources, such as mammal and most preferably be primate or tool pawl animal origin, obtain egg mother cell such as ox;
(ⅲ) make described oocyte enucleation;
(ⅳ) by merging or injecting human or animal's cell or consideration convey are moved in the non-nucleus egg mother cell of the animal kind that is different from donorcells or nuclear;
(ⅴ) the NT product of cultivation gained or NT unit are to produce multi-cellular structure; With
(ⅵ) cultivation derives from described embryo's cell to obtain blastocyte or stem cell-like cell and stem cell-like cell colony.
Consideration convey moves technology or nuclear transfer technology discloses in the literature and is described in a lot of lists of references of mentioning in the background of invention. Example is seen Campbell etc., Theriogenology, 43:181 (1995); Collas etc., Mol.Report Dev., 38:264-267 (1994); Keefer etc., biological self reproducing, 50:935-939 (1994); Sims etc., Proc.Natl. Acad.Sci., USA, 90:6143-6147 (1993); WO94/26884; WO94/24274 and WO90/03432 (listing this paper in as a reference all in full). In addition, United States Patent (USP) 4,944 has been described the method for nuclear transplantation in cattle in 384 and 5,057,420, and the method is also seen Cibelli etc., science, Vol.280:1256-1258 (1998).
Can obtain and cultivate human or animal's cell by well-known method, be preferably mammalian cell. Can be used for humans and animals cell of the present invention and comprise for example epithelial cell, nerve cell, epidermal cell, horn cell, hematopoietic cell, melanocyte, the cartilage cell, lymphocyte (B and T lymphocyte), other immunocyte, red blood cell, macrophage, melanocyte, monocyte, fibroblast, cardiac muscle cell and other myocyte etc. In addition, the people's cell that moves for consideration convey can derive from Different Organs, such as skin, and lung, pancreas, liver, stomach, intestines, heart, reproductive organs, bladder, kidney, urethra and other urinary organ etc. These are the example of suitable donorcells. Suitably donorcells namely can be used for any cell or organ that cell of the present invention can derive from health, and it comprises all body cells or reproduction cell. Preferably, donorcells or endorse contains active division, i.e. the cell of non-dormancy is because it is reported that can strengthen like this clone renders a service. In addition, also preferred this donorcells is in the G1 cell cycle.
According to Thomson etc., science, 282:1145-1147 (1998) and Thomson etc., Proc.Natl.Acad.Sci., the cultural method of USA 92:7544-7848 (1995) (listing this paper in as a reference all in full) report can use the blastocyte of gained to obtain embryonic stem cell line.
Can be used as example that consideration convey moves the cell of donor and be the epithelial cell that derives from people's donor oral cavity and become human keratinized cell. Yet as discussed above, disclosed method also is applicable to other people's cell or nuclear. In addition, nucleus can derive from human body cell and reproduction cell.
Also can before moving, consideration convey use proper technology known in the art that donorcells was stagnated in the mitotic division stage.The method that will end at different steps the cell cycle is described in detail in United States Patent (USP) 5,262,409 (listing this paper in as a reference).Specifically, although it is reported that cycloheximide is to mitotic division inhibited (Bowen and Wilson (1995) J.Heredity45:3-9), but handle when itself and electricimpulse and to unite when using, still can be used for improving activation (Yang etc. (1992) to sophisticated ox ovule parent cell, biological self reproducing, 42 (Suppl.1): 117).
The zygote gene activation is relevant with the super acetylize of histone H 4.The same with other compound, system is dripped rhzomorph-A can be with reversible mode inhibition of histone deacetylase (Adenot etc., in 1-cell mouse embryo's protokaryon, the H4 acetylize of male parent and maternal chromatinic otherness has precedence over dna replication dna and differential transcriptional activity, grow (1997,11) 124 (22): 4615-4625; Yoshida etc., Atrichostatin A and trapoxin: survey the new chemical probe (1995,5) 17 (5) of acetylation of histone: 423-430) to the effect of chromatin Structure and function.
For example, it is believed that butyric acid also can cause the super acetylize of histone by the inhibition of histone deacetylase.As if in general, butyric acid is the energy the modification of gene expression, as if under nearly all situation, adding butyric acid in cultured cells can cell growth inhibiting.Butyric acid purposes in this regard is described in United States Patent (USP) 5,681,718 (listing this paper in as a reference).Therefore, before merging, donorcells can be exposed in Atrichostatin A or the another kind of suitable deacetylase inhibitors, perhaps before the activated gene group, this compound be added in the substratum.
In addition, it is believed that transcription factor is suitably regulated the demethylation that sequence also needs DNA near DNA.Existing people has described complete demethylation (Stein etc., the Mol.Reprod.﹠ Dev.47 (4): 421-429) of DNA from 8 cell stages of pre-implantation embryos to the blastocyst stage.In addition,, can use the dna methylation level in the U-18496 reduction cell, cause transcription factor to be easier to regulate sequence potentially near DNA according to (1997) such as Jaenisch.Therefore, can be before fusion donorcells be exposed to U-18496 (5-Aza), or substratum, adds 5-Aza from 8 cell stages to the blastocyst stage.Perhaps, also can use other known method to realize the demethylation of DNA.
Be used for the ovocyte that consideration convey moves and derive from the animal that comprises Mammals and amphibian animal.The suitable Mammals source of ovocyte comprises sheep, ox, sheep, pig, horse, rabbit, goat, cavy, mouse, hamster, rat, primate, people etc.In preferred embodiments, ovocyte derives from primate or tool pawl animal, as ox.
The method of separating ovocyte is well-known in the art.In fact, this method comprises from Mammals or amphibian animal, as separating ovocyte in the ovary of ox or the reproductive tract.The source that bovine oocyte is easy to obtain is the slaughterhouse.
In order successfully to use such as genetically engineered, the technology that consideration convey moves and clones generally must be before the recipient cell that ovocyte is moved as consideration convey, or is fertilized with before developing into the embryo, at the external oocyte maturation that makes at ovocyte and spermoblast.The ovocyte that this method generally need be collected from animal ovary (as the ox ovary that obtains in the slaughterhouse) immature (I in early stage), and before fertilization or stoning, in maturation medium, make oocyte maturation reach the II stage in mid-term until ovocyte, for bovine oocyte, this stage generally occurs in after the sucking-off about 18 to 24 hours.To be called " ripening stage " during this period of time among the present invention.For computing time, " sucking-off " used herein refers to the jejune ovocyte of sucking-off from ovarian follicle.
In addition, consideration convey moves the ovocyte that has successfully used the sophisticated in vivo II stage in mid-term in the technology.In fact, can be by surgical operation collection II ovocyte in sophisticated mid-term in 35 to 48 hours super ovulation or superovulated milk cow or the heifer body after begin to oestrus back or injection human chorionic gonadotropin (hCG) or similar hormone.
It is reported that stoning and consideration convey make the success significant (example see Prather etc., differentiation, 48:1-8,1991) of oocyte maturation to the NT method when moving.In general, successful in the past cloned mammalian embryo practice uses the ovocyte in II stage in mid-term as the acceptor ovocyte, because it is believed that in this stage, ovocyte can by or be enough to be " activated " to handle the nuclear of importing as the sperm of handling fertilization.To domestic animal, Niu Eryan especially, generally after sucking-off about 16-52 hour Activation of Oocyte phase, preferably about 28-42 hour.
For example, can be by Seshagine etc., biological self reproducing, 40,544-606,1989 describedly wash jejune ovocyte with HEPES buffered hamster embryo culture medium (HECM), then in 39 ℃, ovocyte is placed several maturation medium, this substratum is made up of the tissue culture medium (TCM) (TCM) 199 that 50 μ l contain 10% foetal calf serum, wherein also contain suitable gonadotropin,, covered one deck light weight paraffin or silicon on the described substratum as lutropin (LH) and prolan a (FSH) and estradiol.
After time in the changeless ripening stage, generally be about 10 to 40 hours, after preferably being about 16 to 18 hours, make the ovocyte stoning.Before stoning, preferably take out ovocyte, and before taking out a pile cell, be placed among the HECM that contains 1mg Unidasa/ml.By repeatedly moving liquid or can realize this purpose by vortex momently with the very thin transfer pipet in hole.Screen the polar body of the ovocyte of peeling off then, select II ovocyte in mid-term, be used for consideration convey then and move by the existence mensuration of polar body.Carry out stoning by following.
By as United States Patent (USP) 4,994, the described currently known methods of 384 (listing this paper in as a reference) can carry out stoning.For example, with mid-term the II ovocyte place the optional HECM stoning immediately that contains 7.5 μ g Cytochalasin B/ml, perhaps described ovocyte is placed contain 10% rutting sedson cow serum appropriate culture medium, in CRlaa, carry out stoning subsequently again, after preferably being no more than 24 hours, more preferably after 16 to 18 hours, carry out stoning.
Use microsurgical technique, remove the purpose that polar body and contiguous kytoplasm can be realized stoning, screen then to identify by successful non-nucleus egg mother cell with micropipette.By dyeing ovocyte to screen with 1 μ g, 33342 Hoechst dyestuff/ml HECM, observe ovocyte down in UV-irradiation (being less than 10 seconds) then, successful non-nucleus egg mother cell is placed appropriate culture medium.
In the present invention, preferably after maturation in vitro begins about 10 hours to about 40 hours, more preferably after maturation in vitro begins about 16 hours to about 24 hours, most preferably after beginning, maturation in vitro made the stoning of acceptor ovocyte in about 16 to 18 hours.
Then with general allos in the ovum week crack that the single animal or human's cell or the nucleus of enucleation oocyte is transferred to enucleation oocyte, to be used to produce NT unit.Can use animal or human's cell or nucleus and non-nucleus egg mother cell to produce NT unit according to methods known in the art.For example, can merge fused cell by electricity.Be enough to cause the electricimpulse of plasma membrane instantaneous breakdown can finish the electricity fusion by providing.The puncture of plasma membrane is very of short duration, because film can form apace again.In fact, if the film of two adjacency by induced breakdown, by forming the double-layer of lipoid mixture again, the passage aisle between two cells can be opened.Because the unstable of little opening on thermokinetics, it can enlarge until two cells and become a cell.The further discussion of relevant this method can be with reference to the United States Patent (USP) 4,997,384 (listing this paper in as a reference in full) of Prather etc.Can use multiple electricity to merge medium, comprise for example sucrose, mannitol, Sorbitol Powder and phosphate buffer soln.Also can use Sendai virus to merge (Graham, Wister Inot.Symp.Monogr., 9,19,1969) as fusogen.
(less as donor nuclei) in some cases preferably will be examined direct injection to ovocyte rather than use electroporation and merge.This technology is disclosed in Collas and Barnes, Mol.Reprod.Dev., 38:264-267 (1994) (listing this paper in as a reference in full).
Preferably, ovocyte begin ripe after about 24 hours, by using about 15 microseconds of electricimpulse of 90-120V, electricity fusion human or animal's cell and ovocyte in the groove of 500 μ m.After the fusion, the NT unit that gained is merged places appropriate culture medium until activation, as the activation of hereinafter being identified.Generally very fast can the activation generally after being less than 24 hours, preferably can be activated after 4 to 9 hours.
Can activate NT unit by currently known methods, described method for example comprises being lower than cultivates NT unit under the physiological temp, come down to NT unit is implemented cold, or in fact cool temperature shock.But realize this purpose by at room temperature cultivating NT unit most convenient ground, because room temperature is lower than the physiological temp condition of the normal exposure of embryo.
Perhaps, use known activator to activate.For example, confirmed that sperm penetrates the ovocyte that ovocyte can activate pre-fusion in fertilization process, after consideration convey moves, produced the gestation calf identical of the more work of more number with a plurality of genes.Also can be used for activating NT embryo after the fusion such as electricity and the processing of chemical shock or cycloheximide processing.Suitable activation of oocytes method is the theme of the United States Patent (USP) 5,496,720 (listing this paper in as a reference) of Susko-Parrish etc.
For example, by simultaneously or carry out the following step successively and can activate ovocyte:
(ⅰ) increase in the ovocyte the divalent cation level and
(ⅱ) phosphorylation of the cell protein in the reduction ovocyte.
By with ionophoric form, with divalent cation, as magnesium, strontium generally can be realized above-mentioned purpose in the kytoplasm of barium or calcium importing ovocyte.Other method that increases the divalent cation level comprises the use electroshock, and Ethanol Treatment and cage type sequestrant are handled.
Can reduce phosphorylation by currently known methods, for example by adding kinase inhibitor, as the serine-threonine kinase inhibitor, as 6-dimethyl-amino-purine, Staurosporine, 2-aminopurine and sphingosine.
Perhaps, by with Phosphoric acid esterase, import proteinic phosphorylation also capable of inhibiting cell in the ovocyte as Phosphoric acid esterase 2A and Phosphoric acid esterase 2B.
Can in suitable in-vitro culture medium, cultivate activated NT unit until producing protoblast or stem cell-like cell and cell colony.It is well-known in the art being suitable for cultivating and making the sophisticated substratum of embryo.The example that can be used for cultivating and keep ox embryo's known substratum comprises Ham ' sF-10+10% foetal calf serum (FCS), tissue culture medium (TCM)-199 (TCM-199)+10% foetal calf serum, Tyrodes-albumin-lactic acid salt-pyruvate salt (TALP), Dulbecco ' s phosphate buffered saline (PBS) (PBS), Eagle ' s and Whitten ' s substratum.Being used to collect ovocyte and making its sophisticated the most frequently used substratum is TCM-199, and wherein 1 to 20% serum of Tian Jiaing comprises foetal calf serum, newborn infant's serum, the cow serum in rutting sedson, lamb serum or bullock serum.Preferably keeping substratum comprises and contains Earl salt, 10% foetal calf serum, the TCM-199 of 0.2MM Ma pyruvate salt and 50 μ g/ml gentamicin sulphates.Above-mentioned any substratum also comprises the common cultivation with the various kinds of cell type, described cell such as granulosa cell, oviduct cell, BRL cell, uterine cell and STO cell.
Especially, the endometrial epithelial cell of people can implanted early stage and implantation phase secretion leukaemia inhibitory factor (LIF).Therefore, adding LIF is most important for the ectogenesis that strengthens the reconstruct embryo in substratum.Use LIF to be described in United States Patent (USP) 5,712,156 (listing this paper in as a reference) for protoblast or stem cell-like cell culture.
Another kind is kept substratum and is described in Rosenkrans, the United States Patent (USP) 5,096,822 (listing this paper in as a reference) of Jr etc.This embryo culture medium that is called as CR1 contains the necessary nutritive substance of embryo support.CR1 contains L-lactic acid half calcium, and its amount is preferably 1.0mM to 5.0mM for 1.0mM to 10mM.L-lactic acid half calcium is the L-lactic acid salt that wherein is mixed with half calcium salt.
The appropriate culture medium of keeping the person embryo cell in cultivation is described in Thomson etc., science 282:1145-1147 (1998) and Proc.Natl.Acad.Sci., USA 92:7844-7848 (1995).
Subsequently, preferably with the one or more NT units of appropriate culture medium washing through cultivating, then cell is placed this substratum, described substratum such as CRIaa substratum, Ham ' s F-10, tissue culture medium (TCM)-199 (TCM-199), Tyrodes-albumin-lactic acid salt-pyruvate salt (TALP), Dulbecco ' s phosphate buffered saline (PBS) (PBS), Eagle ' s or Whitten ' s substratum also contain the 10%FCS that has an appointment in the preferred above-mentioned substratum.Preferably in containing the suitable orifice plate that is paved with feeder layer, cultivate.Suitable feeder layer comprises for example inoblast and epithelial cell, as derives from inoblast and the uterine epithelial cell of tool pawl animal, chick fibroblast, mouse (as mouse or rat) inoblast, STO and SI-m220 feeder cell system and BRL cell.
In preferred embodiments, feeder cell contain mouse embryo fibroblasts.The method for preparing suitable inoblast feeder layer is described in hereinafter among the embodiment, and is that those skilled in the art are well-known.
On feeder layer, cultivate NT unit is suitable for obtaining can be used for producing embryonic stem cell like cell or cell colony until the size of NT unit cell.Preferably these NT units are cultured to size and are at least about 2 to 400 cells, be more preferably 4 to 128 cells, most preferably be at least about 50 cells.Can be under proper condition, promptly at about 38.5 ℃ and 5%CO
2In cultivate, replaceable substratum during cultivation is so that the growth optimization generally every 2 to 5 days, was preferably changed 1 subculture approximately every 3 days.
NT unit for the bovine oocyte of end user's cell/stoning obtains obtained being enough to produce the cell of ES cell colony in 12 days after beginning to activate ovocyte, generally be about 50 cells.Yet, along with as the specific cells of nuclear donor, the kind of specific ovocyte and culture condition different, above-mentioned situation can be different.Those skilled in the art are easy to determine when the cell that obtains required enough numbers by naked eyes according to the morphology of the NT unit through cultivating.
People/people's consideration convey is moved the embryo, and it is favourable using the known substratum that can be used for keeping the people's cell in the tissue culture.The example that is applicable to cultivator embryo's substratum comprises Jones etc., the human reproduction., 13 (1): the substratum of 169-177 (1998) report, the P1-catalog number (Cat.No.) is that (these two kinds of substratum all can derive from Irvine Scientific for the substratum of #99242 and substratum that the P-1 catalog number (Cat.No.) is #99292, Santa Ana, and (1998) and (1995) (document is the same) used substratum such as Thomson California).
As mentioned above, the used cell of the present invention preferably includes mammalian somatic cell, most preferably comprises the cell of the mammalian cell cultures that derives from active propagation (non-dormancy).In particularly preferred embodiments, general by adding, lack or replace required dna sequence dna and donorcells is carried out genetic modification.For example, availablely provide required gene product, the DNA construct transfection of expressing as therapeutical peptide or be converted and come from for example people, the donorcells of primate or ox is as keratinocyte or inoblast.The example of described therapeutical peptide comprises lymphokine, as the IGF-I, and IGF-II, Interferon, rabbit, G CFS, reticular tissue polypeptide (as collagen), gene, thrombin, enzyme, enzyme inhibitors etc.
As mentioned above, before consideration convey moves, can modify to obtain other required effect, for example destroy cell lineage and grow, strengthen fetal development and/or suppress apoptosis donorcells.Hereinafter the example of desirable modification will be discussed further.
Therefore one aspect of the present invention comprises donorcells, carries out genetic modification so that there is defective in its pedigree as people's cell, can not produce the offspring of survival when being used for consideration convey and moving.This moves the embryo for people's consideration convey and is particularly useful, because for ethical reason, the embryo who produces survival is not the result that people want.Genetic engineering modified by people's cell is carried out, specific cell lineage can not be divided into when making it be used for consideration convey and move and above-mentioned purpose can be realized.Specifically, but pair cell carries out genetic modification, so that when this cell moved donor as consideration convey, gained " embryo " did not contain or lack basically mesoderm, at least one in entoderm or the ectoderm tissue.
Hope is by knocking out or destroy one or more mesoderms, and the expression of entoderm or ectoderm specific gene realizes above-mentioned purpose, and the example of described gene comprises:
Mesoderm: SRF, MESP-1, HNF-4, β-l integrin, MSD;
Entoderm: GATA-6, GATA-4;
Ectoderm: rna helicase enzyme A, H β 58.
What list above is to participate in mesoderm, the non-limitative example of the known of entoderm and ectoderm growth.Reported in the document in the past that mesoderm is damaged, damaged and damaged cell of ectoderm of entoderm and embryo's generation.Example is seen Arsenian etc., EMBO J., Vol.17 (2): 6289-6299 (1998); Saga Y, Mech.Dev., Vol.75 (1-2): 53-66 (1998); Holdener etc. grow, and Vol1 120 (5): 1355-1346 (1994); Chen etc., Genes Dev.Vol.8 (20): 2466-2477 (1994); Rohwedel etc., developmental biology, 201 (2): 167-189 (1998) (mesoderm); Morrisey etc., Genes Dev.Vol.12 (22): 3579-3590 (1998); Soudais etc. grow Vol.121 (11): 3877-3888 (1995) (entoderm); With Lee etc., Proc.Natl.Acad.Sci.USA, Vol.95:(23): 13709-13713 (1998); With Radice etc., grow Vol.111 (3): 801-811 (1991) (ectoderm).
In general, can be to the desired body cell, as human keratinized cell, epithelial cell or inoblast carry out genetic engineering modified so that one or more genes that are specific to the specific cells pedigree " are knocked out " and/or described expression of gene is significantly weakened.By currently known methods, reorganization can realize this purpose as homology." knock out " the used preferred gene system of required gene and be disclosed in the United States Patent (USP) 5,631,153 and 5,464,764 of Capecchi etc., just wherein reporting-bearing selection (PNS) carrier, the dna sequence dna of this carrier in can the required mammalian genes group of targeting modification.This genetic modification can cause cell can not be divided into specific cell lineage when moving donor as consideration convey.
Can be used for producing pedigree-damaged consideration convey through the cell of genetic modification and move the embryo, promptly this embryo can not produce at least one functional mesoderm, entoderm or ectoderm.Therefore, even the gained embryo is for example implanted in the people uterus, also can not produce the offspring of survival.Yet the ES cell that moves generation by this consideration convey is still useful, because they can produce the cell of one or two residual not ruined pedigree.For example, people's consideration convey that ectoderm is damaged moves the embryo and still can produce and derive from mesoderm and endoblastic noble cells.By the damaged cell of one or two produced ectoderm in disappearance and/or destruction rna helicase enzyme A or H β 58 genes.
Also can carry out genetic modification to express another kind of required dna sequence dna to the damaged donorcells of these pedigrees.
Therefore, can produce the damaged blastocyst of pedigree through the donorcells of genetic modification, when the pawnshop was long all one's life, multipotency was divided into two germ layers.
Perhaps, donorcells can be modified so that its " death ".Can realize this purpose by antisence DNA or ribozyme telomerase gene.By known engineering method that antisense DNA or ribozyme express being provided or can realizing this purpose by gene knockout.When being used for consideration convey and moving, these " death " cells can not be divided into the offspring of survival.
Another preferred embodiment of the present invention is to produce to move the embryo by the consideration convey of more effective growth in tissue culture.It why favourable reason to be it should be able to reduce produces ES cell and/or offspring's (if blastocyst is implanted female surrogate) required time and essential fusion.Why desirable Another reason is for it: according to observations, blastocyst and ES cell that consideration convey moves generation have weakened developmental potentiality.Although often can alleviate these problems by changing conditions of tissue culture, another kind of terms of settlement is to strengthen fetal development by strengthening the expression of gene that participates in fetal development.
For example, it is reported that MHC I family member Ped type gene product is extremely important to fetal development.More particularly, it is reported that for the mouse pre-implantation embryos, Q7 and Q9 gene are responsible for " growth fast " phenotype.Therefore, hope will provide these and genes involved, or the DNA of the expression of its people or other Mammals counterpart import donorcells can produce growth more fast consideration convey move the embryo.With respect to moving for the embryo by merging consideration convey that allogenic cell or nucleus produce, the embryo that consideration convey moves generation between xenogenesis grows slowlyer in tissue culture, and therefore, aforesaid method is particularly useful to it.
The DNA construct importing donor somatocyte that especially, can before consideration convey moves, will contain Q7 and/or Q9 gene.For example, but the construction expression construct, it contains with Q7 and/or Q9 gene can operate the strong composing type mammalian promoter that links to each other, IRES, and (as Xin Meisu, ADA is DHFR) with the poly-A-sequence, as bGH polyA sequence for one or more appropriate selection marks.In addition, further strengthen Q7 by comprising spacer (insulate) and the Q9 expression of gene also is favourable.Hope can be at these genes of early expression of blastocyst growth, because these genes in (as ox, goat, pig, dog, cat and people) not of the same race are high conservatives.Also wish donorcells is transformed other gene that can strengthen fetal development to insert.Therefore, these donorcellses through genetic modification should be able to more effectively produce blastocyst and implant embryo in earlier stage.
Another aspect of the present invention comprises structure pair cell apoptosis, and promptly apoptosis has the donorcells of resistance.According to the literature, the gene relevant with necrocytosis is present in (Adams etc., science, 281 (5381): 1322-1326 (1998)) among the embryo who implants early stage.It is reported that but the gene of cell death inducing comprises for example Bad, Bok, BH3, Bik, Hrk, BNIP3, BimL, Bad, Bid and EGL-1.On the contrary, it is reported that the gene that can protect cell to avoid apoptosis for example comprises: BcL-XL, Bcl-w, Mcl-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K and CED-9.
Therefore, can make up donorcells, wherein the gene of cell death inducing " is knocked out " or is wherein protected cell to avoid apoptotic expression of gene to be enhanced in embryo development procedure or to open.
For example, can realize this purpose by importing DNA construct, this construct can provide above-mentioned protective gene in embryo development procedure, as the regulating and expressing of Bcl-2 or genes involved.Therefore, get final product " unlatching " gene by under specific growth conditions, cultivating the embryo.Perhaps, also said gene can be linked to each other with constitutive promoter.
More particularly, can make up the promotor that contains with adjustable or composing type (as PGK, SV40, CMV, ubiquitin or beta-actin), IRES, suitably selective marker and the poly-A sequence DNA construct that can operate the Bcl-2 gene that links to each other, and the donor mammalian cell that its importing is required, in human keratinized cell or inoblast.
When these donorcellses were used to produce consideration convey and move the embryo, they should have resistance by the pair cell apoptosis, thereby can more effectively differentiation in tissue culture, thereby the speed and/or the number that are moved the suitable pre-implantation embryos of generation by consideration convey increase to some extent.
But the another kind of method that can obtain identical result is to destroy one or more expression of gene of cell death inducing.By knocking out these genes or can realizing this purpose by using at antisense DNA or ribozyme at the gene of fetal development early expression and cell death inducing, its example sees above.Perhaps, can make up donorcells, make it contain two kinds of modifications, i.e. the gene of energy of rupture cell death inducing and enhancing can stop or prevent the expression of gene of apoptosis.The clone that influences the structure of gene of apoptosis and selection and expressing said gene is disclosed in the invention people for Craig B.Thompson etc., the artificial close United States Patent (USP) 5,646,008 (listing this paper in as a reference) of holding root university of assigning.
Strengthen a kind of method that consideration convey moves effectiveness and be that to select to be in the cell in the specific cells phase of the cycles be donorcells.It is reported that this moves to render a service to consideration convey remarkably influenced (Barnes etc., Mol.Reprod.Devel., 36 (1): 33-41 (1993)).Selection is in the existing people's report of different methods of the cell in the specific cells phase of the cycles, and described method comprises serum starvation method (Campbell etc., nature, 380:64-66 (1996); Wilmut etc., nature, 385:810-813 (1997)) and chemical synchronization (Urbani etc., Exp.Cell Res., 219 (1): 159-168 (1995)).For example, but can be with specific cyclin DNA and adjusting sequence and mark note, can operate continuously as green fluorescent protein (GFP), the back connects cyclin again and destroys box, and optional being connected with isolated sequence to strengthen the expression of cyclin and labelled protein.Take this, be easy to the naked eye detect and select the cell of required cell cycle and move donor used as consideration convey.For example, in order to select to be in the cell of G1 phase, need to use the cyclin D1 gene.Yet any cyclin gene all should be applicable to the present invention's (example is seen King etc., cellular elements biology, Vol.7 (9): 1343-1357 (1996)).
Yet, need be with invasive less or more efficient methods produce the cell that is in required cell cycle phase.Hope makes it express specific cyclin under the condition that can survey and realizes above-mentioned purpose by donorcells is carried out genetic modification.Take this, be easy to from other cell cycle, tell the cell in specific cells cycle.
Cyclin is only in specific cell cycle phase expressed protein.They comprise the cyclin D1 of G1 phase, D2 and D3, cyclin E, A and the H of the cell periodic protein B 1 of G2/M phase and B2 and S phase.These protein are easy to be translated in cytogolcytosol and destroy.Described proteinic " instantaneous " expresses the existence that part gives the credit to " destruction box ", this destroys box is short aminoacid sequence, be to can be used as marker destroys above-mentioned cyclin rapidly via the ubiquitin approach with mediation a proteinic part (Adams etc., science, 281 (5321): 1322-1326 (1998)).
In the present invention, can make up donorcells, make its under the condition that is easy to detect (preferred naked eyes as seen, as by using fluorescent mark) express one or more above-mentioned cyclin genes.For example, but can be with specific cyclin DNA and adjusting sequence and mark note, can operate continuously as green fluorescent protein (GFP), the back connects cyclin again and destroys box, and optional being connected with isolated sequence to strengthen the expression of cyclin and/or labelled protein.Take this, be easy to the naked eye detect and select the cell of required cell cycle and move donor used as consideration convey.For example, in order to select to be in the cell of G1 phase, need to use the cyclin D1 gene.Yet any cyclin gene all should be applicable to the present invention's (example is seen King etc., cellular elements biology, Vol.7 (9): 1343-1357 (1996)).
Another aspect of the present invention is to strengthen the method that consideration convey moves effectiveness, is preferably to strengthen the method that consideration convey between xenogenesis moves effectiveness.Although the inventor has illustrated the consideration convey that can obtain producing blastocyst when the nucleus of a kind or cell being inserted or being blended in the enucleation oocyte of another kind and moved the embryo, described embryo can produce ES clone, and the effectiveness of this method is very low.Therefore, generally need merge many times and produce blastocyst, the cell of cultivating described blastocyst could produce ES cell and ES clone.
The ectogenetic method that another kind of enhancing consideration convey moves the embryo is to make the culture condition optimization.A method that obtains this result is to stop cultivation NT embryo under the apoptotic condition.About this embodiment of the present invention, (example is seen Jurisicosva etc. to have found proteolytic enzyme such as capsases can to cause ovocyte death by the apoptosis that is similar to other cell type, Mol.Reprod.Devel., 51 (3): 243-253 (1998)).
Hope is by adding the growth that one or more capsase inhibitor strengthen blastocyst being used for the substratum that consideration convey moved and kept blastocyst or cultivate to implant the embryo in early stage.Described inhibitor comprises for example capsase-4 inhibitors I, the capsase-3 inhibitors I, capsase-6 inhibitor II, capsase-9 inhibitor II and capsase-1 inhibitors I, its amount is effectively to suppress apoptotic amount, as accounts for 0.00001 to 5.0% of substratum weight; More preferably account for 0.01% to 1.0% of substratum weight.Therefore, can use aforesaid method, improve the effectiveness that consideration convey moves by strengthening blastocyst and embryo's growth in tissue culture subsequently.
Obtain after the NT unit of required size, from district's band (zone), take out cell, be used to produce protoblast or stem cell-like cell and clone then with apparatus.Preferably realize this purpose through the following steps, promptly take out the cell mass that contains NT unit, it generally contains at least about 50 cells, washs this cell, and cell is laid on feeder layer, as on the inoblast of irradiation.Must the hang oneself internal layer overwhelming majority of the NT unit of cultivating that the cell that is used to obtain stem cell-like cell or cell colony is general, its size is preferably at least 50 cells.Yet NT unit that cell number is less or bigger and the cell that derives from NT unit's other parts also can be used for obtaining ES-like cell and cell colony.
Donorcells DNA is exposed the process that the long period will help dedifferenting in the cytosol of ovocyte.In this regard, by from the embryo of reconstruct, taking out blastomere and itself and new non-nucleus egg mother cell fusion can being realized cloning again.Perhaps, donorcells and non-nucleus egg mother cell can be merged, after 4 to 6 hours, need not to activate, take out karyomit(e) and merge, activate again subsequently with more young ovocyte.
At suitable growth medium, as be added with in the feeder layer among the α MEM of 10%FCS and 0.1mm beta-mercaptoethanol (Sigma) and L-glutaminate and keep cell.Often change growth medium on demand so that the growth optimization was changed a subculture according to appointment every 2 to 3 days.
This cultural method can form protoblast or stem cell-like cell or clone.For the NT embryo of derived from human cell/bovine oocyte, in α MEM substratum, cultivate about colony that promptly can be observed more than 1 day.Yet, along with specific nuclear donor cell, specific ovocyte and culture condition different, incubation time is also different.Those skilled in the art can change culture condition where necessary so that the growth optimization of specific protoblast or stem cell-like cell.
The outward appearance that gained protoblast or stem cell-like cell and cell colony show with as the kind of karyocyte donor rather than similar as the protoblast or the stem cell-like cell of the kind of donor ovocyte.For example, for for protoblast or stem cell-like cell that people's nuclear donor cell transfer to the bovine oocyte of stoning is obtained, the form that cell reveals more is similar to mouse embryo stem cell rather than ox ES-like cell.
More particularly, more at large do not define each cell that people ES-is a cell colony as yet, the periphery of described colony can reflect and smooth in appearance.In addition, the doubling time of cell colony is longer, is about the twice of mouse ES cells.With derived from the ES cell of ox and pig different be that described colony does not have epithelial cell sample outward appearance.
As mentioned above, according to Thomson at United States Patent (USP) 5,843, the report in 780, primate stem cell is SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) and alkaline phosphatase (+).Hope can show similar or identical marker representation according to the people that the inventive method produces with primate ES cell.
Perhaps, can produce all mesoderms according to cell, the ability of ectoderm and entoderm tissue confirms that this cell is people or primate embryonic stem cells really.Can illustrate this point by the ES cell of cultivating the present invention's generation under proper condition, described culture condition is disclosed in for example United States Patent (USP) 5,843,780 (listing this paper in as a reference) of Thomson.
The protoblast of gained or stem cell-like cell and clone are preferably person embryo cell or stem cell-like cell and clone and have multiple treatment and diagnostic use.Most particularly, described protoblast or stem cell-like cell can be used for the Transplanted cells therapy.Person embryo cell or stem cell-like cell can be used for treating multiple disease.
In this regard, known mouse embryonic stem (ES) cell almost can be divided into any cell type, as hemopoietic stem cell.Therefore, the person embryo cell or the stem cell-like cell of the present invention's generation should have similar differentiation capability.Can induce protoblast of the present invention or stem cell-like cell to obtain required cell type according to currently known methods with differentiation.For example, by in division culture medium and under the condition that allows cytodifferentiation, cultivating protoblast of the present invention or stem cell-like cell, can induce its differentiation to produce hemopoietic stem cell, the myocyte, myocardial cell, liver cell, the chondrocyte, epithelial cell, urethra cell etc.Cause the substratum of embryonic stem cell differentiation and the currently known methods that method is this area, suitable culture condition also is well-known in the art.
For example, Palacios etc., Proc.Natl.Acad.Sci., USA, 92:7530-7537 (1995) discloses by stem cell is induced and has produced hemopoietic stem cell by embryonic lineage, described revulsion comprises: culturing stem cells aggregate in lacking the suspension culture base of vitamin A acid originally, then in containing the same medium of vitamin A acid, cultivate, and then cell aggregation is transferred in the substrate that cell attachment can be provided.
In addition, Pedersen.J.Reprod.Fertil.Dev., 6:543-552 (1994) is one piece of summary, the many pieces of papers of mentioning in this article disclose the vitro differentiation embryonic stem cell to produce the differentiation of multiple differentiated cell types, described cell type comprises hematopoietic cell, the myocyte, myocardial cell, neurocyte etc.
In addition, Bain etc., developmental biology, 168:342-357 (1995) discloses the vitro differentiation embryonic stem cell and has had the method for the neurocyte of neuron behavior with generation.These reference be reported obtain the example of the method for noble cells by protoblast or stem cell-like cell.These reference, especially wherein disclosed relevant differentiating embryonic is all listed this paper in as a reference in full in the content of the method for cell.
Therefore, those skilled in the art use currently known methods and substratum can cultivate protoblast of the present invention or stem cell-like cell to obtain required differentiated cell types, as neurocyte, and myocyte, hematopoietic cell etc.
In addition, using derivable Bcl-2 or Bcl-xl may be useful to the ectogenesis that strengthens the specific cells pedigree.In vivo, Bcl-2 can prevent a lot (but not being whole) the apoptosis type necrocytosiss in lymph and the neurodevelopment process.United States Patent (USP) 5,646 has gone through after the transfection donorcells in 008 (the listing this paper in as a reference), and how the expression with Bcl-2 is used for suppressing relevant cytophyletic apoptosis.
Can use protoblast of the present invention or stem cell-like cell to obtain any required differentiated cell types.The therepic use of people's cell of this differentiation is impayable.For example, but in needing the therapeutic process of bone marrow transplantation end user's hemopoietic stem cell.Can use this method to treat a variety of diseases, terminal cancer (ovarian cancer and leukemia) and jeopardize immune disease for example is as AIDS.By for example with cancer or AIDS patient's adult body cell, as epithelial cell or lymphocyte and non-nucleus egg mother cell, merge as bovine oocyte, obtain protoblast mentioned above or stem cell-like cell, and under the condition that helps breaking up, cultivate this cell until obtaining hemopoietic stem cell, take this to obtain hemopoietic stem cell.Can use this hemopoietic stem cell treatment to comprise the disease of cancer and AIDS.
Perhaps, neuropath's the adult body cell and the animal ovocyte of stoning can be merged as primate or bovine oocyte, therefrom obtain person embryo cell or stem cell-like cell, under differentiation condition, cultivate this cell to produce neuronal cell line.For example comprise by transplanting the treatable specified disease of this human nerve cell: Parkinson's disease, presenile dementia, ALS and cerebral palsy etc.Specific to Parkinson's disease, the fetal brain neurocyte of having illustrated implantation can be set up with cell on every side and good get in touch and can produce Dopamine HCL, so just can reverse symptoms of Parkinson's Disease for a long time.
For specificity is selected noble cells, available selective marker transfection donorcells of expressing via inducible promoter is when inducing the cell lineage that differentiation phase can be selected or enrichment is specific.For example, can use CD34-neo to select hemopoietic stem cell, Pw1-neo selects the myocyte, and Mash-1-neo selects sympathetic neuron, and Mal-neo selects the CNS neurone of people's pallium grey matter etc.
Of the present invention one big advantage is to supply gene such as grade or the isogenic people's cell that is suitable for transplanting in fact endlessly.Therefore, can avoid the present serious problems that implantation method brought, promptly owing to host versus graft or graft versus host repel the rejection to transplanted tissue that takes place.Usually, can prevent or alleviate rejection by the anti-rejection drugs of using such as S-Neoral.Yet described medicine has remarkable deleterious side effect, as immunosuppression, and carcinogenic nature and very expensive.The present invention should be able to eliminate or reduce demand to anti-rejection drugs at least greatly.
Comprise for example Spinal injury Deng treatable other disease of gene cell therapy and illness, multiple sclerosis, muscular dystrophy, diabetes, hepatopathy, i.e. hypercholesterolemia, heart trouble, cartilage is replaced, burn, ulcer of foot, gastrointestinal disorder, vascular disease, ephrosis, urinary tract is sick and increase diseases associated and illness with the age.
Person embryo cell or stem cell-like cell that the present invention produces can be used for producing through genetic engineering modified or genetically modified people's noble cells.In fact, by importing required one or more genes (described gene can be a heterologous gene), or remove the person embryo cell of the present invention's generation or all or part of endogenous gene of stem cell-like cell, make this cytodifferentiation become required cell type to achieve the above object.The preferred method that obtains this modification is by homologous recombination, because can use this technology to insert, and one or more genes at one or more specific sites place of disappearance or modified stem cell like cell genome.
Can use this methodology to replace damaged gene, for example damaged immunity system gene, the cystic fibrosis gene, or cause having the gene of the protein expression of therapeutic action, described protein such as somatomedin, lymphokine, cytokine, enzyme etc.For example, the gene that coding can be derived from the somatomedin of brain imports person embryo cell or stem cell-like cell, and the cytodifferentiation neuroblast is implanted this cell the loss that can stop neurocyte in the ill process in parkinsonian's body.
In the past, being had nothing in common with each other from the primary cell to the immortal cell line by BDNF cells transfected type, can be to derive from neural cell, also can not be to derive from neural cell (sarcoplast and inoblast).For example, can use retroviral vector BDNF gene transfection stellate cell, and cell is implanted (Yoshi-moto etc., brain research, 691:25-36 (1995)) in the parkinsonian rat model.
Therapy can be at the back 32 days Parkinson's disease with rat-sample sxs 45% of transfer in this one.In addition, the casein '-hydroxylase gene is placed stellate cell also can obtain similar result (Lundberg etc., Develop.Neurol., 139:39-53 (1996) and the reference of wherein mentioning).
Yet system also has problems in this body.Especially, the retroviral vector that uses is reduced in vivo at present, and transgenosis only can transient expression (Mulligan, science, 260:926-932 (1993)).And, the primary cell that the research is used, the stellate cell life-span is limited, duplicates slowly.These characteristics have disadvantageous effect to transfection speed, and have hindered the selection to the cell of stable transfection.In addition, may breed the used big gene target primary cell colony of homologous recombination technique hardly.
On the contrary, should be able to eliminate the problem relevant by end user's protoblast or stem cell-like cell with the retrovirus system.Transferee of the present invention had illustrated the embryonic lineage of ox and pig in the past can be transfected and select to be used for the stable integration allogeneic dna sequence DNA.This method also is described in commonly assigned U.S.'s serial number 08/626,054 (listing this paper in as a reference in full) of submitting on April 1st, 1996.Therefore, use this method or other known method required gene can be imported in person embryo cell of the present invention or the stem cell-like cell, cytodifferentiation becomes required cell type, as hematopoietic cell, and neurocyte, pancreatic cell, chondrocyte etc.
The gene that can import person embryo cell of the present invention or stem cell-like cell comprises for example Urogastron, Prostatropin, derive from neuroglial neurotrophic growth factor, Regular Insulin-like growth factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine gene (interleukin-, Interferon, rabbit, G CFS, tumour necrosis factor (α and β), Deng), the curative enzyme of encoding, collagen, the gene of human serum albumin etc.
In addition, also can use the present known negative selective system in this area to remove patient's therapeutic cell in case of necessity.For example, can be caused producing the protoblast that contains the TK gene by the donorcells of thymidine kinase (TK) gene transfection.The differentiation of these cells can cause also expressing the separation of the required therapeutic cell of TK gene.By use 9-(1,3-dihydroxy-2-third oxygen methyl) guanine can be at any time in patient's body selective clearing therapeutic cell.Should negative selective system be described in United States Patent (USP) 5,698,446 (listing this paper in as a reference).
Protoblast of the present invention or stem cell-like cell preferably also can be used as the vitro differentiation model for people's cell, participate in regulating the gene of early development in particular for research.
The noble cells tissue and the organ that use protoblast of the present invention or stem cell-like cell to obtain also can be used for drug research.
In addition, protoblast of the present invention or stem cell-like cell can be used as nuclear donor to produce other protoblast or stem cell-like cell and cell colony.
In order more clearly to describe the present invention, the spy provides the following example.
Embodiment 1 material and method consideration convey move used donorcells
Scrape gently from the one-tenth human oral inwall of being ready to accept to test with the glass slide of standard and to get epithelial cell.Cell washed from slide contain phosphate buffered saline (PBS) but do not contain the culture dish of Ca or Mg.Repeat to draw cell with the aperture transfer pipet, thereby cell mass is dispersed as single-cell suspension liquid.Then with cell transfer to the TL-HEPES substratum droplet that contains 10% foetal calf serum (FCS) that is coated with oil reservoir, carry out consideration convey and move to be used for bovine oocyte to stoning.The consideration convey shifting method
Basic consideration convey shifting method was once described in the past.Briefly, after the oocyte maturation in slaughterhouse, from a pile cell, peel off ovocyte external make, and after maturation about 18 hours (hpm) with the micropipette stoning of bevel.Containing bisbenzimide (Hoechst 33342,3 μ g/ml; Sigma) confirm stoning in the TL-HEPES substratum.Then each donorcells is placed in all cracks of ovum of acceptor ovocyte.The electricity consumption integration technology is merged the kytoplasm and the donor nuclei (NT unit) of bovine oocyte.NT unit is used the fusion pulse of once forming by 90V 15 microseconds.This step begins ripe back 24 hours (hpm) at ovocyte and carries out.Place the CRlaa substratum until 28hpm NT unit.
The method that is used for artificial activation's ovocyte has been described in other places.NT unit activates in 28hpm and carries out.Being briefly described as follows of Activiation method: NT unit is exposed to ionomycin (the 5 μ M among the TL-HEPES that is added with 1mg/ml BSA; CalBiochem, La Jolla CA) reaches 4 minutes, then with the TL-HEPES washing that is added with 30mg/ml BSA 5 minutes.Again NT unit is transferred in the CRlaa substratum droplet that contains 0.2mM DMAP (Sigma), in 38.5 ℃, 5%CO
2The middle cultivation 4 to 5 hours.Washing NT unit is placed among the CRlaa substratum+10%FCS+6mg/ml BSA in 4 well culture plates that contain the mouse embryo fibroblasts feeder layer (will be described below) that is paved with then.In 38.5 ℃, 5%CO
2In NT unit was cultivated more than 3 days.Changed a subculture every 3 days until active period till the 12nd day.At this moment, reach required cell number with apparatus taking-up from band, the NT unit of promptly about 50 cell numbers, and be used to produce embryonic lineage.Photo by the above-mentioned NT unit that obtains is shown in Fig. 1.The inoblast feeder layer
The primary culture of embryo fibroblast derives from the tire mouse in 14 to 16 day age.Aseptic taking-up head, liver, after heart and the digestive tube, the embryo is cut with scissors broken, in 37 ℃ of trypsinase EDTA solution (0.05% trypsinase/0.02%EDTA in pre-temperature; GIBCO, Grand Island, NY) middle insulation is 30 minutes.Inoblast is laid in the tissue culture flasks, is being added with 10% foetal calf serum (FCS) (Hyclone, Logen, UT), α-MEM the substratum of penicillin (100IU/ml) and Streptomycin sulphate (50 μ l/ml) (BioWhittaker, Walkersville, MD) the middle cultivation.Went down to posterity irradiation 35 * 10Nunc culture dish (Baxter Scientific, McGaw Park, IL) embryo fibroblast in back 3 to 4 days.In 37 ℃, containing 5%CO
2Damp atmosphere in cultivate and keep inoblast through irradiation.Use then and have the culture plate culturing embryo clone of homogeneous cell monolayer.Produce embryonic lineage
Washing directly is laid on it on the raising inoblast of irradiation by the above-mentioned NT unit cell that obtains.These cells comprise the interior layer segment of NT unit.In the growth medium of forming by the α MEM that is added with 10%FCS and 0.1mM β mercaptoethanol (Sigma), keep cell, changed a growth medium every 2 to 3 days.Cultivate and promptly can be observed initial colony more than 1 day.Colony is constantly bred, and shows and the similar form of previously disclosed mouse embryonic stem (ES) cell.More at large do not define each cell in the colony as yet, the periphery of described colony can reflect and smooth in appearance.In addition, the doubling time of cell colony slow than mouse ES cells.With derived from the ES cell of ox and pig different be that described colony does not still have epithelial cell sample outward appearance so far.Fig. 2 to 5 is the photos by the above-mentioned ES like cell colony that obtains.Produce people's cell of differentiation
The person embryo cell of gained is transferred in the division culture medium, and cultivates until the people's cell type that obtains breaking up.The result
Table 1. is donor nuclei in the generation of NT unit with in growing with people's cell
| Cell type | NT unit's number of preparation | NT unit's number (%) of 2 cell stages | 4 numbers (%) to the NT unit of 16 cell stages | NT unit's number (%) of 16 to 400 cell stages |
| Lymphocyte | 18 | ?12(67％) | ????3(17％) | ????0 |
| Mouth epithelial cells | 34 | ?18(53％) | ????3(9％) | ????1(3％) |
| Adult fibroblasts | 46 | ?4(9％) | 12 (4 cells; 26%) 8 (8-16 cells; 17.4%) | ????--- |
Be laid on the inoblast feeder layer developing into a NT unit that has greater than the structure of 16 cells.This structure is attached to feeder layer, and begins to breed the colony (example is seen Fig. 2) that formation has ES cell sample form.In addition, although do not attempt using the structure of 4 to 16 cell stages to produce the ES cell colony, forefathers have proved that this stage can produce ES or ES like cell system (mouse, Eistetter etc., Devel.Growth and Differ., 31:275-282 (1989); Ox, Stice etc., 1996).Therefore, the expection 4 NT units to 16 cell stages should also can produce protoblast or stem cell-like cell and cell colony.
Clone by will becoming human keratinized cell with containing ACM, uridine, the stoning bovine oocyte of cultivating in the substratum of glucose and 1000IU LIF merges also can obtain similar result.In the embryo of 50 reconstruct, have 22 cleaved, have 1 to develop into blastocyst at about the 12nd day, this blastocyst is paved, promptly begin to produce ES clone.
Although with reference to multiple specific material, method and embodiment describe and have illustrated the present invention, should understand the present invention and be not limited to specific material, combination of materials and selected for this reason method.Multiple so detailed variation those skilled in the art will envision that and understands.
Claims (50)
1. produce the method for protoblast or stem cell-like cell, described method comprises the following steps: that (ⅰ) is being suitable for forming under the condition that consideration convey moves (NT) unit, the people of required differentiation or mammalian cell or nucleus are inserted in the animal ovocyte of stoning, and wherein said ovocyte derives from the animal kind different with people or mammalian cell; (ⅱ) nuclear transplantation unit of activation gained; (ⅲ) cultivate described through the activated nuclear transplantation unit until greater than 2 cell development stages; (ⅳ) cultivation derives from the cell of described NT unit through cultivating to obtain protoblast or stem cell-like cell.
2. the process of claim 1 wherein that the cell that inserts stoning animal ovocyte is people's cell.
3. the method for claim 2, wherein said people's cell is an adult cell.
4. the method for claim 2, wherein said people's cell is an epithelial cell, keratinocyte, lymphocyte or inoblast.
5. the method for claim 2, wherein ovocyte derives from Mammals.
6. the method for claim 5, wherein the animal ovocyte derives from tool pawl animal.
7. the method for claim 6, wherein said tool pawl animal is selected from ox, sheep, pig, horse, dog and wild ox.
8. the process of claim 1 wherein that enucleation oocyte is ripe before stoning.
9. the process of claim 1 wherein that the nuclear transplantation unit that merges is activated external.
10. the process of claim 1 wherein and on the feeder layer culture, cultivate the activated nuclear transplantation unit.
11. the method for claim 10, wherein feeder layer contains inoblast.
12. the step that the process of claim 1 wherein (ⅳ) is to cultivate the cell have in 16 or the more cellulous NT unit on feeder layer.
13. the method for claim 12, wherein said feeder layer contains inoblast.
14. the method for claim 13, wherein said inoblast contains mouse embryo fibroblasts.
15. the process of claim 1 wherein that gained protoblast or stem cell-like cell can break up through inducing.
16. the method for claim 2, wherein gained protoblast or stem cell-like cell can break up through inducing.
17. the process of claim 1 wherein to merge and merge realization by electricity.
18. the protoblast or the stem cell-like cell that obtain according to the method for claim 1.
19. the person embryo cell or the stem cell-like cell that obtain according to the method for claim 2.
20. the person embryo cell or the stem cell-like cell that obtain according to the method for claim 3.
21. the person embryo cell or the stem cell-like cell that obtain according to the method for claim 4.
22. the person embryo cell or the stem cell-like cell that obtain according to the method for claim 6.
23. the person embryo cell or the stem cell-like cell that obtain according to the method for claim 7.
24. people's cell of the differentiation that the method by claim 16 obtains.
25. people's cell of the differentiation of claim 24, it is selected from neurocyte, hematopoietic cell, pancreatic cell, myocyte, chondrocyte, urethra cell, liver cell, splenocyte, sexual cell, skin cells, intestinal cells and gastric cells.
26. methods of treatment, described method comprise people's cell of using the homogenic differentiation of claim 24 to the patient who needs the Transplanted cells therapy.
27. the method for claim 26 is wherein implemented medicable disease of described Transplanted cells therapy or illness and is selected from Parkinson's disease, Huntington, presenile dementia, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatopathy, diabetes, heart trouble, cartilage defects or damage, burn, ulcer of foot, vascular disease, urinary tract disease, AIDS and cancer.
28. the method for claim 26, wherein Fen Hua people's cell is hematopoietic cell or neurocyte.
29. the method for claim 26, methods of treatment wherein can be treated Parkinson's disease, and the cell of differentiation is a neurocyte.
30. the method for claim 26, methods of treatment wherein can be treated cancer, and the cell of differentiation is a hematopoietic cell.
31. people's cell of the differentiation of claim 24, it contains and expresses the gene of insertion.
32. the process of claim 1 wherein and in described protoblast or stem cell-like cell, insert, remove or modify required gene.
33. the method for claim 32, wherein required genes encoding curative enzyme, somatomedin or cytokine.
34. the method for claim 32, wherein said protoblast or stem cell-like cell are person embryo cell or stem cell-like cell.
35. the method for claim 32 is wherein removed by homologous recombination, modifies or lack required gene.
36. the process of claim 1 wherein donorcells is carried out genetic engineering modified to destroy entoderm, the growth of at least one in ectoderm and the mesoderm.
37. the process of claim 1 wherein that donorcells is carried out genetic modification to be renderd a service to strengthen differentiation.
38. the method for claim 36 is wherein cultivated the nuclear transplantation unit through cultivating in the substratum that contains at least a capsase inhibitor.
39. the process of claim 1 wherein that but the donorcells expression can demonstrate the mark note that the specific cells cyclin is expressed.
40. the method for claim 36, wherein donorcells has changed after modified and has been selected from following expression of gene: SRF, MESP-1, HNF-4, β-1 integrin, MSD, GATA-6, GATA-4, rna helicase enzyme A and H β 58.
41. the method for claim 37 is wherein carried out genetic modification to import the DNA that Q7 and/or Q9 genetic expression can be provided to described donorcells.
42. can operating with adjustable promotor, the method for claim 41, wherein said gene link to each other.
43. the process of claim 1 wherein donorcells apoptosis capable of inhibiting cell behind genetic modification.
44. the method for claim 43 wherein is selected from the apoptosis that following one or more expression of gene can provide minimizing by change: Bad, Bok, BH3, Bik, Blk, Hrk, BNIP3, GimL, Bid, EGL-1, Bcl-XL, Bcl-w, Mcl-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bcl-2, E1B-19K and CED-9.
45. the method for claim 44, wherein at least one described gene can be operated with inducible promoter and link to each other.
46. mammalian somatic cell, but the DNA of described cell expressing coding mark note, but the expression of described mark note links to each other with the specific cells cyclin.
47. the cell of claim 46, wherein cyclin is selected from cyclin D1, D2, D3, B1, B2, E, A and H.
48. the cell of claim 46, but wherein the mark note is a fluorescent polypeptide.
49. the cell of claim 48, wherein said mammalian cell is selected from the people, primate, rodent, tool pawl animal, dog and cat cell.
50. the cell of claim 48, wherein said cell is the people, the cell of ox or primate.
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| CN113249297A (en) * | 2014-05-28 | 2021-08-13 | 儿童医院医疗中心 | Methods and systems for converting precursor cells to stomach tissue via directed differentiation |
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| MXPA02002744A (en) * | 1999-09-14 | 2003-07-21 | Univ Massachusetts | Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions. |
| BR0014864A (en) * | 1999-10-15 | 2002-11-19 | Advanced Cell Tech Inc | Methods of producing differentiated progenitor cells and defective embryonic stem cells in lineage |
| CN1425064A (en) * | 1999-12-20 | 2003-06-18 | 马萨诸塞大学 | Embryonic or stem-like cells produced by cross species nuclear transplantation |
| US6921665B2 (en) | 2000-11-27 | 2005-07-26 | Roslin Institute (Edinburgh) | Selective antibody targeting of undifferentiated stem cells |
| US6576464B2 (en) * | 2000-11-27 | 2003-06-10 | Geron Corporation | Methods for providing differentiated stem cells |
| AU2002250020A1 (en) * | 2001-02-02 | 2002-08-19 | Erik Forsberg | Method of cloning transgenic mammalian animals using pseudonuclei |
| CA2466203A1 (en) * | 2001-11-06 | 2003-05-15 | Shanghai Second Medical University | Cross-species somatic cell nuclear transfer using rabbit oocyte |
| CA2466198A1 (en) | 2001-11-06 | 2003-05-15 | Shanghai Second Medical University | Somatic cell derived embryonic stem cells and their differentiated progenies |
| AU2003230725A1 (en) * | 2002-04-01 | 2003-10-20 | Gtc Biotherapeutics, Inc. | A method for selecting cell lines to be used for nuclear transfer in mammalian species |
| US10865383B2 (en) | 2011-07-12 | 2020-12-15 | Lineage Cell Therapeutics, Inc. | Methods and formulations for orthopedic cell therapy |
| WO2013177228A1 (en) | 2012-05-22 | 2013-11-28 | Loma Linda University | Generation of integration/transgene-free stem cells |
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| US5905042A (en) * | 1996-04-01 | 1999-05-18 | University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus | Cultured inner cell mass cell lines derived from bovine or porcine embryos |
| CA2262817A1 (en) * | 1996-08-19 | 1998-02-26 | James Robl | Embryonic or stem-like cell lines produced by cross species nuclear transplantation |
| BR9808192A (en) * | 1997-03-06 | 2000-10-24 | Infigen Inc | Animal cloning process |
| AU8587598A (en) * | 1997-07-26 | 1999-02-16 | Wisconsin Alumni Research Foundation | Trans-species nuclear transfer |
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| CN113249297A (en) * | 2014-05-28 | 2021-08-13 | 儿童医院医疗中心 | Methods and systems for converting precursor cells to stomach tissue via directed differentiation |
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| EP1060243A1 (en) | 2000-12-20 |
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