CN1265599A - Cloning using donor nuclei from non-serum starved, differentiated cells - Google Patents

Cloning using donor nuclei from non-serum starved, differentiated cells Download PDF

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CN1265599A
CN1265599A CN98807691A CN98807691A CN1265599A CN 1265599 A CN1265599 A CN 1265599A CN 98807691 A CN98807691 A CN 98807691A CN 98807691 A CN98807691 A CN 98807691A CN 1265599 A CN1265599 A CN 1265599A
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cell
disease
nucleus
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S·L·司蒂斯
J·西柏利
J·M·罗贝尔
P·戈吕克
F·A·彭斯迪莱昂
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University of Massachusetts UMass
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Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei from non-serum starved cells into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Description

Use is cloned from the donorcells nuclear of non-serum starved noble cells
Invention field
The present invention relates to go into from the nuclear transplantation mammal of the mammalian cell of non-serum starved, differentiation cloning process with donorcells nuclear phase enucleation mammal ovocyte of the same race.This nucleus is carried out reprogram to instruct clone's fetal development, it can be implanted into female receptor then to produce fetus or offspring or to be used to produce the inner cell mass cell (CICM) of cultivation.This clone's embryo also can combine with the embryo of fertilization to produce chimeric embryo, fetus and/or offspring.
Background of invention
Existing report uses inner cell mass (ICM) cell of ungulate to carry out nuclear transplantation.For example, the mature oocyte that discloses in Mol.Reprod.Dev.38:264-267 (1994) by cracked donorcells microinjection being gone into enucleation such as Collas carries out the nuclear transplantation of cattle ICMs.Collas etc. disclose wherein four-head pregnancy and two are produced.And Keefer etc. are at Biol.Reprod., when disclosing in the nuclear transplantation method blastocyte implantation that the ICM cell with cattle produces as donor nuclei among the 50:935-939 (1994) and going into the receptor of cattle, have produced some offsprings that can survive.And Sims etc. report in institute of NAS and disclose among the 90:6143-6147 (1993) by will producing calf at the mature oocyte that the cattle ICM of external short term culture nucleus is transferred to enucleation.
Can produce the lamb (Campbell etc., nature, 380:64-68 (1996)) that can survive behind the placenta cells nuclear that also has the transplanting of reporting to cultivate.Further, generation (Stice etc., Biol.Reprod, the 54:100-110 (1996) of paotoblastic use of cattle multipotency and chimeric fetus in the nuclear transplantation of existing report; Collas etc., Mol.Reprod.Dev., 38:264-267 (1994)).Proof granular cells such as Collas (one-tenth somatic cell) can be used for the cattle cloning process to produce the embryo.But, fail to prove early stage (blastocyst stage) growth afterwards of embryo.And granular cell is difficult for cultivating and only can be from female acquisition.Collas etc. do not attempt breeding granular cell or attempt those cells of genetic modification in cultivation.Wilmut etc. (nature, 365:810-813 (1997)) produce nuclear transplantation sheep offspring from fetal fibroblast, and have produced an offspring from adult sheep cell.
In the field that produces transgenic pig, also have problems.By existing method, allogeneic dna sequence DNA is introduced in early embryo or the embryonic lineage that is divided into various cell types in the fetus and finally develops into transgenic animal.But producing transgenic animal needs many early embryos, so this method is unusual poor efficiency.And, the simple and effective method of screening transgenic embryo before the female gonosome of agency put into embryo by spended time and funds not.In addition, the gene targeting technology is not easy to finish with the early embryo transgenic technology.
Embryonic stem cell in the mice makes research worker can screen transgenic cell and carries out gene targeting.This causes comparing and will using genetic engineering more with other transgenic technology.But, embryonic stem cell line and other embryonic lineage must be maintained undifferentiated state, this needs feeder layer and/or add cytokine in culture medium.Even taked these preventive measures, spontaneous differentiation still can often take place and can not produce transgenic progeny by existing obtainable method in these cells.And some embryonic lineages must be bred in the mode that is unfavorable for the gene targeting method.
The method of dried (ES) cell line of external acquisition embryo is well known from the previous pre-mice embryo of transplanting.(see Evans etc. for example, nature, 29:154-156 (1981); Martin, institute of NAS newspaper, 78:7634-7638 (1981)).If because fibroblastic feeder layer (Evans etc., Id.) or differentiation inhibition source (Smith etc., biology progress, 121:1-9 (1987)) exist, the ES cell is gone down to posterity with undifferentiated state.
Reported in the past that the ES cell had many application.For example, reported that the ES cell can be used as the external model of differentiation, be particularly useful for studying the gene that relates in the early development adjusting.When mouse ES cells being introduced the mice embryo of pre-transplanting, can produce germline mosaic, thereby prove their versatility (Bradley etc., nature, 309:255-256 (1984)).
Because the ES cell is delivered to follow-on ability with its genome, they have the potential use that the ES cell that has or do not have a required genetic modification by use carries out the operation of livestock animals kind system.And, livestock animals if any the hoof animal in, come to transplant in advance freely growth (Smith etc., Biol.Reprod., 40:1027-1035 (1989) that endorsing of domestic animal embryo promotes enucleation oocyte; With Keefer etc., Biol.Reprod., 50:935-939 (1994)).On the contrary, it is reported that the nuclear that surpasses the embryo of 8 cell stages from mice does not promote the growth (Cheong etc., Biol.Reprod., 48:958 (1993)) of enucleation oocyte after transfer.Therefore, be very ideal from the ES cell of livestock animals, because they can provide through the potential source of the totipotency donor nuclei of genetic manipulation or be used for the nuclear transplantation method.
Some research groups have been reported the separation that is called the versatility embryonic lineage.For example, Notarianni etc. are at J.Reprod.Fert.Suppl., reported the foundation from the stable totipotent cell of thinking of pig and sheep blastocyst system among the 43:255-260 (1991), this cell line shows some and similar morphology and growth characteristics of cell from the primary culture of the isolating inner cell mass of sheep blastocyst immunosurgery.Simultaneously, Notarianni etc. are also at J.Reprod.Fert.Suppl., and disclosing among the 41:51-56 (1990) from inferring of pig blastocyst is the keeping and break up of culture of totipotency embryonic lineage.Gerfen etc. are in Animal Biotechnology, and 6 (1): disclose among the 1-14 (1995) from the pig blastocyst and separated embryonic lineage.These cells can be in the fibroblast feeder layer of the mice embryo of service condition culture medium not stable maintenance, and it is reported and in incubation, be divided into several different cell types.
And Saito etc. are at Roux ' s Arch.Dev.Biol., the cattle embryo thousand cell like cells that report is cultivated among the 201:134-141 (1992) three generations of can surviving, but dead the 4th back of going down to posterity.Handyside etc. disclose the inner cell mass of cultivating the isolating sheep embryo of immunosurgery under the condition of separation from the mouse ES cells system of mice ICMs at Roux ' s Arch.Dev.Biol. among the 196:185-190 (1987).Handyside etc. are reported under such condition, and sheep ICMs adheres to, spreads the zone of the concurrent ES of bringing out cell sample and endoderm cell's like cell, but only have the entoderm like cell obviously as seen after the cultivation of carrying out the longer time.
Recently, Cherny etc. are at Theriogenology, have reported among the 41:175 (1994) that the genitaloid cell line of versatility cattle of calling oneself can keep in the long-term cultivation process.These cells after cultivating about 7 days, produce the positive ES sample colony of alkali phosphatase (AP) dyeing, and performance forms the ability of embryoid, and spontaneously are divided at least two kinds of different cell types.It is reported that these cells also express the mRNA of transcription factor OCT4, OCT6 and HES1, this be sure of it is specially by the pattern of the homeobox gene of ES cellular expression.
And, recent Campbell etc. are at nature, reported among the 380:64-68 (1996) comfortable in the future promote to cultivate under the isolating condition of ES cell line in the mice 9 day age the sheep embryo Placenta Hominis (ED) cell of cultivation carry out can producing the lamb of survival after the nuclear transplantation.The author reaches a conclusion, and the ED cell of sheep embryo was totipotent after the process nuclear transplantation from 9 day age, and this totipotency can be kept in incubation.
Van Stekelenburg-Hamers etc. have reported separation that is called permanent cell line and CHARACTERISTICS IDENTIFICATION from cattle blastocyst inner cell mass cell at Mol.Reprod.Dev. among the 40:444-454 (1995).The author under different condition, separate and cultivate from the ICM of the cattle blastocyst in 8 or 9 day age with measure any accessory cell and culture medium promote cattle ICM cell adhere to and grow in the most effective.They reach a conclusion adhering to of the ICM cell cultivated and grow and can be enhanced by the culture medium of using the additional serum (rather than standard serum) with activated carbon-desorbing of STO (l cell) accessory cell (the intrauterine chrotoplast that replaces cattle) and use.Yet Van Stekelenburg etc. has reported that their cell line more is similar to epithelial cell rather than versatility ICM cell.
Smith equals disclosed WO 94/24274 on October 27th, 1994, Evans and equals April 5 nineteen ninety disclosed WO 90/03432 and equal on November 24th, 1994 disclosed WO 94/26889 with Wheeler and reported and think the separating of the stem cell animal that can be used for obtaining transgenic animal, screening and breeding.Evans etc. also reported from pig and cattle species think to produce transgenic animal useful be called deriving of multipotent stem cells.And Wheeler equals on November 24th, 1994 disclosed WO 94/26884 and also discloses and think to preparing the chimeric embryonic stem cell useful with genetically modified ungulate.
Therefore, based on foregoing content, can obviously find out many groups because ES cell line in clone or the generation of transgenic embryo and the potential application in the nuclear transplantation, has attempted preparing ES cell line.
Although the existing report of former document still needs improving one's methods of cloning mammal.
Purpose of the invention and overview
New and the improved method that the purpose of this invention is to provide the mammal (as embryo, fetus and offspring) that is used to produce the clone.
The present invention's purpose more specifically provides the new method of the nuclear transplantation of mammalian cell non-serum starved, differentiation being gone into enucleation oocyte mutually of the same race that relates to that is used for cloning mammal.
Another object of the present invention provides the method that is used to breed the Adult Mammals with certified hereditary superiority or other ideal character.
Another object of the present invention provides and is used to produce improving one's methods of genetic engineering or genetically modified mammal (being embryo, fetus, offspring).The present invention also provides genetic engineering or the genetically modified mammal that produces by this method.
Purpose more specifically of the present invention provides by inserting, remove or modify the target DNA sequence in mammalian cell that is breaking up before cell that uses differentiation or the nucleus formation NT unit or nucleus and produces genetic engineering or genetically modified mammiferous method.The present invention also provides genetic engineering or the genetically modified mammal that produces in this way.
Another object of the present invention provides by the nuclear transplantation of the cell of non-serum starved, genetically modified, differentiation being gone into noble cells enucleated oocyte mutually of the same race and produces genetic engineering or genetically modified mammiferous method.The present invention also provides genetic engineering or the genetically modified mammal that produces in this way.
Another object of the present invention provides to relate to goes into the new method that produces mammal CICM cell with noble cells enucleation oocyte mutually of the same race with the nuclear transplantation of the cell of non-serum starved, differentiation.
Another object of the present invention provides the CICM cell that produces with the cell that breaks up enucleation oocyte mutually of the same race by the nuclear transplantation of the mammalian cell of non-serum starved, differentiation is gone into.
The present invention's purpose more specifically provides and relates to the method that the human oocyte that becomes somatic nuclear transplantation to go into enucleation non-serum starved people's cell such as people produces people CICM cell.
Another object of the present invention is to use such CICM cell to be used for the treatment of or diagnoses.
Specific purposes of the present invention be to use such CICM cell comprise the CICM cell of people and ungulate be used for the treatment of or diagnose any wherein cell, tissue or organ transplantation the treatment on or the diagnosis on useful disease.These CICM cells can use in mutually of the same race or cenospecies.
Another object of the present invention is to use and derives from the NT embryo, fetus or offspring's cell or tissue comprises that people and ungulate tissue are used for the treatment of or diagnose any wherein cell, the tissue or organ transplantation the treatment on or the diagnosis on useful i or I comprise parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic disease, heart disease, the cartilage displacement, burn, angiopathy, urethral disease and be used for immunodeficiency, bone marrow transplantation, other treatment of diseases such as cancer.These tissues can use these tissues in mutually of the same race or cenospecies.
Another specific purposes of the present invention are to use the CICM cell that produces according to the present invention to be used to produce cell, tissue or the organ of differentiation.
The present invention's purpose more specifically is to use the people CICM cell that produces according to the present invention to be used to produce people's cell, tissue or the organ of differentiation.
Another specific purposes of the present invention CICM cell that to be external uses produce according to the present invention for example is used for the research of cell differentiation and test objective as being used for drug research.
Another object of the present invention provides improving one's methods of transplantation therapy, comprises the application that waits gene or homogenic cell, tissue or organ that produces from the CICM cell that produces according to the present invention.Such Therapeutic Method comprises that disease for example and damage comprise other treatment of diseases such as the treatment of parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic disease, heart disease, cartilage displacement, burn, angiopathy, urethral disease and immunodeficiency, bone marrow transplantation, cancer.
Another object of the present invention provides by insert, remove or modify genetic engineering or the genetically modified CICM cell that the target DNA sequence produces in mammalian cell that is breaking up before cell that uses this differentiation or the nucleus formation NT unit or nucleus.
Another object of the present invention is to use the transgenic or the genetic engineering CICM cell that produce according to the present invention to be used for gene therapy, to be particularly useful for treating and/or preventing of above-mentioned disease and damage.
Another object of the present invention is to use the CICM cell that produces according to the present invention or transgenic or engineered CICM cell as the nucleus donor that is used for nuclear transplantation.
Therefore, on the one hand, the invention provides the method that is used for cloning mammal (as embryo, fetus, offspring).Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(iii) the NT unit with described cultivation is implanted into host mammal so that NT unit develops into fetus.
Preferably, cultivating activatory nuclear moves and grows unit until surpassing the 2-cell development stage.
The cell of these fetuses, tissue and/or organ can be advantageously used in cell, tissue and/or filed of organ transplantation.
The present invention also comprises by inserting, remove or modified the target DNA sequence and clone genetic engineering or genetically modified mammiferous method in the mammalian cell of differentiation or nucleus before mammalian cell that will differentiation or nucleus inserting enucleation oocyte.
The present invention also provides mammal and those the mammiferous offsprings that obtain according to the method described above.
The present invention is preferably used for cloning ungulate.
On the other hand, the invention provides the method that produces the CICM cell.Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(i) activate the nuclear transplantation unit that obtains; And
(iii) cultivate the cell that obtains from the NT unit of described cultivation to obtain the CICM cell.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The CICM cell can be advantageously used in cell, tissue and filed of organ transplantation.
About above-mentioned and hereinafter more obviously other purposes of the present invention, advantage and characteristics, feature of the present invention can more be expressly understood by detailed description and the additional claim with reference to the following preferred embodiment of the invention.Detailed Description Of The Invention
The invention provides by nuclear transplantation or consideration convey and move improving one's methods of cloning mammal.Among the application, consideration convey moves or nuclear transplantation or NT are used interchangeably.
According to the present invention, will from the nuclear transplantation of the mammalian cell of non-serum starved, differentiation go into donorcells nuclear phase enucleation mammal ovocyte of the same race in.Pair cell nuclear carries out reprogram to instruct clone's fetal development, it can be implanted into female receptor then to produce fetus and offspring or to be used to produce the CICM cell.Clone's embryo also can combine with the embryo of fertilization to produce chimeric embryo, fetus and/or offspring.
The method of prior art has been used the blastocyte type in cloning process.This comprises the work of (Biol.Reprod., 54:100-110,1996) such as Campbell etc. (nature, 380:64-68,1996) and Stice.In two of these researchs, embryonic lineage is less than 10 days embryo from gestation.In these two researchs, cell is kept on feeder layer to prevent the obvious differentiation of the used donorcells of cloning process.Found that the present invention uses fetus or becomes somatic cell effective.
Do not expected that the clone embryos that has fetus or adult donorcells nuclear can grow senior embryo and the fetal state.The science doctrine once was to have only the body early embryo cell type can instruct this type of growth.Do not expected that a large amount of clone embryos can or become somatic cell to produce from the embryo.Also have, it is unexpected that new transgenic embryo cell line can obtain this fact from the transgene clone embryo easily.
One-tenth somatic cell and fetal fibroblast from sheep on purpose are used to produce sheep offspring (Wilmut etc., 1997).Yet in that research, the nucleus donorcells of the serum starvation of emphasizing that is to use resting stage is important for the success of Wilmut cloning process.For the present invention, do not exist such to serum starvation or immobilized requirement.On the contrary, use mammalian cell non-serum starved, differentiation to realize the clone.And according to the present invention, cloning efficiency is same to have nothing to do with using fetus or adult donorcells, and Wilmut etc. (1997) report is lower with the cloning efficiency of adult donorcells.
Therefore, according to the present invention, mammal comprises that the superior genotypic breeding of ungulate is possible.This will allow to have the breeding of the adult animal of certified gene superiority or other ideal character.For example in many important ungulate kinds, will speed up improvement.By the present invention, the countless fetuses that can gather in the crops in cloning process and utilize can be arranged or become somatic cell.This may produce many identical offsprings at short notice.
The method of inferring Wilmut etc. in addition will cause the generation (seeing MacQuitty, Nature Biotech., 15:294 (1997)) of transgenic animal.But, have no reason to infer for example control oneself transfection the somatic nuclear energy of one-tenth of foreign DNA in the nuclear transplantation process, survive.In this, known by manipulation in vitro change mouse embryonic stem (ES) thus the characteristic of cell obtains the ability that their form survival chimeric embryo.Therefore, before the present invention, the clone of transgenic animal does not obtain prediction.
The present invention also can simplify transgenic method by the cell source of energy clonal propagation is operated.This has got rid of the needs that cell maintained undifferentiated state, therefore, comprises that the genetic modification of random integration and gene targeting is easier to realize.The method is also more effective by the nuclear transplantation and the ability of external modification and selection cell are combined the transgenic embryo technology that becomes than former.According to the present invention, these cells can be when the acellular factor, conditioned medium and/or feeder layer clonal propagation, further simplify and help transgenic method.When in cloning process, using transfectional cell, can produce the transgenic embryo that can develop into fetus and offspring according to the present invention.And these genetically modified clone's embryos can be used for producing CICM cell line or other embryonic lineage.Therefore, the present invention has got rid of for helping gene engineering at the external needs of deriving and keeping undifferentiated cell system.
The present invention also can be used for producing CICM cell, fetus or the offspring who can be used for for example cell, tissue and organ transplantation.By getting fetus from animal or become somatic cell and it is used for cloning process, can develop into organ at them and obtain various kinds of cell, tissue and possible organ from clone's fetus in forming.Cell, tissue and organ also can separate from clone's offspring.This method can be provided for " material " source that many medical science and veterinary's therapy comprise cell and gene therapy.If these cell transplantations get back to cell from animal in, so just avoided immunologic rejection.And, because the various kinds of cell type can be from these clone and separate, the chimerism of other method system such as hemopoietic can be used to avoid mutually of the same race in and plant between the immunologic rejection of animal.
Therefore, on the one hand, the invention provides the method for cloning mammal.Usually, mammal produces by the nuclear transplantation method that comprises the following steps:
(i) obtain the source of required mammalian cell non-serum starved, differentiation as donorcells nuclear;
(ii) from the mammal acquisition oocyte mutually of the same race with donorcells nuclear derived cell;
(iii) with described oocyte enucleation;
(iv) for example by merging or injecting required noble cells or nuclear transplantation are gone in the non-nucleus egg mother cell to form NT unit;
(v) activate the NT unit that obtains; And
(vi) the NT unit of described cultivation is moved and grow into host mammal so that NT unit develops into fetus.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The present invention also comprises by the method for inserting, removing in mammalian cell that is breaking up before mammalian cell that will break up or the nucleus insertion enucleation oocyte or nucleus or modification target DNA sequence is cloned genetic engineering or genetically modified animal.
The present invention also provides mammal and those the mammiferous offsprings who obtains according to the method described above.The present invention is preferably used for cloning ungulate.
The present invention further provides the application of NT fetus and NT and chimeric offspring in cell, tissue and filed of organ transplantation.
On the other hand, the invention provides the method that produces the CICM cell.Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(iii) cultivate the cell that obtains from the NT unit of described cultivation to obtain the CICM cell.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The CICM cell is advantageously used in cell, tissue and filed of organ transplantation or is used to produce fetus or the offspring comprises transgenic fetus or offspring.
As used herein, fetus is the unborn immature viviparous animal of taking out from the uterus.Therefore, the period of fetus of cattle is from becoming pregnant back 35 days up to birth.The period of fetus of pig is from becoming pregnant back 30 days up to birth.Mammal is an adult from birth to death.
Preferably, at least 2～400 cell sizes are cultivated by NT unit, 4～128 cells more preferably, and most preferably at least about 50 cell sizes.
Consideration convey moves technology or nuclear transfer technology to be had in the literature in many lists of references of describing and quoting in background of invention description is also arranged.See especially: Campbell etc., Theriogenology, 43:181 (1995); Collas etc., Mol.Report.Dev., 38:264-267 (1994); Keefer etc., Biol.Reprod., 50:935-939 (1994); Sims etc., institute of NAS newspaper, 90:6143-6147 (1993); WO 94/26884; WO 94/24274 and WO90/03432 here quote them as a reference fully.And U.S. Patent number is 4,944,384 and 5,057, and 420 patent has also been described the method for the nuclear transplantation of cattle.
Being meant of differentiation has the characteristics different with on every side structure or primary cell or the cell of function.The mammalian cell of differentiation is that those have passed through the body early embryo stages of cell.More specifically, the cell of differentiation is that those are from the cell that passes through the Placenta Hominis stage (embryogenetic the 10th day of cattle) at least.The cell of differentiation can be from ectoderm, mesoderm or entoderm.
Mammalian cell comprises that people's cell can obtain by well-known method.Can be used for mammalian cell of the present invention and comprise for example cell, fibroblast, myocardial cell and other muscle cell etc. of epithelial cell, neurocyte, epidermis cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, lymphocyte (B and T lymphocyte), erythrocyte, macrophage, mononuclear cell, monokaryon.And the mammalian cell that is used for nuclear transplantation can be from acquisitions such as different organs such as skin, lung, pancreas, liver, stomach, intestinal, heart, genitals, bladder, kidney, urethra and other urinary organss.These only are the examples of suitable donorcells.Suitable donorcells, promptly useful in the present invention cell can obtain from any cell of health or organ.This comprises all somatic cells or sexual cell.
Fibroblast is required cell type, because they can obtain from fetus and the adult animal that grows in large quantities.Fibroblast has differentiation to a certain degree, therefore in the past thinks that they are the relatively poor cell types that are used for cloning process.But importantly be, these cells can external with doubling time fast breed easily and can clonal propagation to be used for the method for gene targeting.The present invention be novel also be because used the cell type of differentiation.The present invention is advantageous, because cell is easy in external breeding, genetic modification and screening.
The cloning process of other report (as Wilmut etc., 1997) depends on the cell of using serum starvation.Yet among the present invention, donorcells is not at the serum starvation state.According to (1997) such as Wilmut, the cell of serum starvation is immobilized,, withdraws from trophophase that is.Additive method (chemistry, temperature etc.) also can produce akinete.Used donorcells is not an akinete among the present invention.
There are sheep, cattle, pig, goat, horse, rabbit, Cavia porcellus, mice, hamster, rat, primate etc. in the suitable mammal source of oocyte.Oocyte is preferably from ungulate, most preferably obtain from cattle.
The method of separating oocyte is well-known in this area.Basically, this comprises ovary or reproductive tract separation oocyte from mammal such as cattle.The source of the bovine oocyte that is easy to obtain is the slaughterhouse material.
Successfully use for making as genetic engineering, nuclear transplantation and clone's technology, general oocyte is as before the recipient cell of nuclear transplantation, and can be by the spermatid fertilization with must be at maturation in vitro before developing into embryo at them.This process generally need be collected immature (I in early stage) oocyte as the cattle ovary that obtains in the slaughterhouse from Mammalian Ovary, and making it ripe until the oocyte arrival II stage in mid-term in maturation medium before fertilization or enucleation, this generally occurs in drew bovine oocyte after about 18-24 hour.To achieve the object of the present invention, this time period is called " period of maturation ".As used herein, for section computation time, " absorption " is meant and draws immature oocyte from ovary follicle.
And, be successfully used in the nuclear transfer technology at the oocyte in external mature II stage in mid-term.Basically, sophisticated II oocyte in mid-term 35 to 48 hours non-super ovulation or superovulated cow or the collection of heifer Chinese and foreign department after rutting period or behind injection human chorionic gonadotropin (hCG) or the similar hormone.
The success of existing maturation period of reporting the oocyte in enucleation and the nuclear transplantation to the NT method is important.(for example seeing Prather etc., differentiation, 48,1-8,1991).Usually, successful mammal embryo cloning process is can or to be enough to obtain the nuclear that sperm that " activation " be fertilized with the picture processing is handled introducing because be sure of oocyte in this stage as the receptor oocyte with the oocyte in II stage in mid-term.In domestic animal, the oocyte pot-life generally is after absorption about 16-52 hour, preferably about 28-42 hour.
For example, can be as Seshagine etc., biology of reproduction 40,544-606, wash immature oocyte in the buffered hamster embryo culture medium of HEPE described in 1989 (HECM), just be placed on then and contain comprising of 50 microlitres of suitable promoting sexual gland hormone such as during the maturation medium of the tissue culture medium (TCM) (TCM) 199 of 10% hyclone of lutropin (LH) and follicle-stimulating hormone (FSH) and estradiol drips under 39 ℃ of thin layer paraffin or the silicon layer.
From about 10-40 hour, and after one section preferably about 16-18 hour fixed maturation time, with the oocyte enucleation.After enucleation, preferred mobile oocyte and before removing the upright cell of ovum, oocyte is placed the HECM of the hyaluronidase that contains 1 milligram every milliliter.This can beat or of short duration rotation is finished by repeating to inhale with the suction pipe of pore very.Then, the polar body of the oocyte that screening is exposed, and according to the existence of polar body and the oocyte of the metaphase of cell division of definite choosing just is used for nuclear transplantation.Enucleation subsequently.
Enucleation can be undertaken by known method, as being described in 4,994,384 the patent at U.S. Patent number, here quotes as a reference.For example, can with mid-term II oocyte place optionally that every milliliter of HECM that contains 7.5 microgram cytochalasin Bs (CB) is used for enucleation immediately, perhaps can place proper culture medium, for example embryo culture medium such as CRlaa, add 10% rutting period Ox blood serum, a little enucleations in evening then, preferably be no more than 24 hours after, and more preferably 16-18 hour.
Available micropipette is removed polar body and peripheral cell matter is finished enucleation with coming microsurgery.Screening and Identification goes out those successful non-nucleus egg mother cells.This screening can be observed oocyte and carry out then by 33342 Hoechst dyeings with every milliliter 1 microgram among the HECM in 10 seconds under ultraviolet radiation.Successful then non-nucleus egg mother cell can place proper culture medium, as adds the CRlaa of 10% serum.
Among the present invention, the preferably ripe beginning back enucleation in about 10-40 hour time outside the people of receptor oocyte more preferably began the back about 16-24 hour at maturation in vitro, and most preferably maturation in vitro began the back about 16-18 hour.
Be transferred the ovum week in the crack that enters the non-nucleus egg mother cell that is used for producing NT unit subsequently with non-nucleus egg mother cell single mammalian cell mutually of the same race.According to methods known in the art, mammalian cell and non-nucleus egg mother cell will be used to produce NT unit.For example, adopt electricity to merge fused cell.Merge by providing the electric pulse that is enough to cause the plasma membrane instantaneous breakdown to finish electricity.Because film is repaired rapidly, plasma membrane punctures very of short duration.Therefore, if two adjacent films by induced breakdown and their lipid bilayer mix repair after, two intercellular passage aisles will be opened.Because the thermodynamic phase of these little openings, it enlarges and becomes one up to two cells.The United States Patent (USP) 4,997,384 of Prather etc. (is incorporated herein by reference at this) as a reference in full is used for further discussing the method.Available multiple electricity merge medium comprise as, sucrose, mannitol, Sorbitol and phosphate buffered solution.Also can use Sendai virus and finish fusion (Graham, Wistor Inot.Symp.Monogr., 9,19,1969) as fusion agent.
And (when for example using less donor nuclei) is injected directly into nuclear that merge than electricity consumption in the oocyte may be more preferred in some cases.This technology is at Collas and Barnes, and Mol.Reprod.Dev. openly, here quotes as a reference among the 38:264-267 (1994) fully.
Preferably, mammalian cell and oocyte oocyte maturation begin the back about 24 hours, merge medium (0.25M D-Sorbitol containing, 100 μ m calcium acetates, 0.5mM magnesium acetate, 1.0g/L BSA (FAF) pH7.2) μ M chambers 500 in, use electric pulse, about 15 microseconds of 90-120V and carry out electricity fusion.After the fusion, the NT unit of the fusion that obtains just places proper culture medium such as Crlaa culture medium until activation.Typical activation is very short time thereafter, typically is less than 24 hours, finishes after preferably about 2～9 hours.
NT unit can activate by known method.These methods comprise, for example, cultivate NT unit at inferior physiological temp, and are cold by using in fact, or the in fact cool cold NT unit of hitting of temperature.Most convenient ground can be by reaching this purpose in incubated at room temperature NT unit, room temperature with respect to embryo the normal physiological temp condition that exposes be colder.
Optionally, can utilize known activating reagent to finish activation.For example, sperm penetrates oocyte and has shown gestation with the multiclass gene consistent calf of pre-activated fusion oocyte to obtain in nuclear transplantation more can survive in fertilization process.Also have, processing method can be used to merging postactivated NT embryo as electricity and chemical shock.The activation method of suitable oocyte is the content of the U.S. Patent number 5,496,720 of Susk-Parrish etc., is incorporated herein by reference in full at this.
In addition, activation also can simultaneously or be carried out subsequently:
(i) level of bivalent cation in the raising oocyte, and
(ii) reduce the phosphorylation of cell protein in the oocyte.This generally can lead to and bivalent cation is imported the oocyte Cytoplasm carries out, as, magnesium, strontium, admire or calcium, as with ionophoric form.The method of other rising bivalent cation levels comprises the application galvanic shock, handles with Ethanol Treatment with the chelating agen of catching.
Can adopt known method to reduce phosphorylation, as, add inhibitors of kinases, as serine-threonine kinase inhibitor such as 6-dimethylamino-purine, D-82041 DEISENHOFEN, 2-aminopurine and sphingol.
Optionally, phosphatase and phosphatase 2A and phosphatase 2B can be imported the phosphorylation that oocyte suppresses cell protein.
In the embodiment, can adopt after fusion in about 24 hours, and in about 2-9 hour the NT unit of merging is exposed in the TL-HEPES culture medium that contains 5 μ M ionomycins and 1mg/mlBSA momently behind preferred the fusion, in the TL-HEPES that contains 30mg/ml BSA, wash the activation of finishing NT subsequently.
Activatory NT unit cultivates subsequently in suitable in-vitro culture medium and produces up to CICM cell and cell colony.Well known embryo culture and the sophisticated culture medium of being suitable for.The example of the known culture medium that can be used for cattle embryo culture and keep comprises that Ham ' s F-10 adds 10% hyclone (FCS), tissue culture medium (TCM)-199 (TCM-199) adds 10% hyclone, Di Luode-albumin-lactate-pyruvate (TALP), Dulbecco ' s phosphate buffered saline (PBS) (PBS), Eagle ' s and Whitten ' s culture medium.Be used for one of oocytes collection and sophisticated prevailing culture medium and be TCM-199 and be supplemented with 1-20% serum comprising hyclone, new cattle calf serum, rutting period cow serum, little sheep blood serum or bull serum.Preferably keep culture medium and comprise the TCM-199 culture medium that contains Earl salt, 10% hyclone, 0.2mM Sodium Pyruvate and 50 μ g/ml gentamycin sulfate.Any one also can participate in the co-cultivation with various kinds of cell type such as granular cell, oviduct cell, BRL cell and uterine cell and STO cell in above-mentioned.
The Roenkrans that is hereby incorporated by has described another kind in the United States Patent (USP) 5,096,822 of Jr etc. and has kept culture medium.The embryo culture medium of this CRl by name contains the necessary nutrient substance of embryo support.
The CRl amount is from 1.0mM to 10mM, and preferred 1.0mM is to L-lactic acid half calcium of 5.0mM.L-lactic acid half calcium is to have half calcium salt L-lactic acid bonded with it.L-lactic acid half calcium is important, mainly asks because a single composition satisfies two of culture medium: (i) be used for closely cytoskeleton and arrange necessary calcium demand; The (ii) necessary lactic acid demand of metabolism and electron transport.L-lactic acid half calcium also can be used as embryo the survive mineral and the energy source of necessary culture medium.
Advantageously, the CRl culture medium does not contain serum, as hyclone, and does not need to use that zooblast is cultivated altogether or the other biological culture medium, promptly contains the culture medium of zooblast such as oviduct cell.Biological medium sometimes may be disadvantageous, and is deleterious and be difficult to the microorganism or the micro-factor that detect, identify and eliminate to the embryo because they may contain.
The example of the main component in the CRl culture medium comprises the bovine serum albumin (sigma A-6003) of L-lactic acid half calcium, sodium chloride, potassium chloride, bicarbonate and micro-FAF.Essential and the non essential amino acid that can in culture medium, add in addition, ormal weight.Containing amino acid whose CRl knows with abbreviation " CRlaa ".
The CRl culture medium preferably contains the following ingredients of following content:
Sodium chloride-114.7mM
Potassium chloride-3.1mM
Sodium bicarbonate-26.2mM
L-lactic acid half calcium-5mM
BSA-the 3mg/ml of fatty acids not
In one embodiment, activatory NT embryo unit was placed the CRlaa culture medium that contains 1.9mM DMAP about 4 hours, in HECM, wash subsequently and in containing the CRlaa of BSA, cultivate then.
For example, activatory NT unit can be transferred in the CRlaa culture medium that contains 2.0mM DMAP (Sigma) and at 38.5 ℃ according to appointment of environmental conditions, 5%CO 2Under cultivated appropriate time 4-5 hour according to appointment.
Afterwards, preferably wash the NT unit of cultivation and with being placed on during proper culture medium cultivates as the CRlaa that contains 10%FCS, and 6mg/ml is included in the orifice plate that preferably contains suitable fusion feeder layer.Suitable feeder layer comprises that for example, fibroblast, STO and the SI-m220 feeder cells of fibroblast of the own hoof animal of penta fibrocyte Tathagata and uterine epithelial cell, chick fibroblast, Mus (as mice or rat) are, and the BRL cell.
In one embodiment, feeder cells comprise mouse embryo fibroblasts.The embodiment that the description of suitable fibroblast feeder layer preparation is seen below and in those skilled in the art, knowing.
At feeder layer (5 * 10 5Cell/ml) is gone up and is cultivated NT unit and reach the size that is suitable for transferring to recipient female up to NT unit, perhaps is suitable for obtaining can be used for producing the size of the cell of CICM cell or cell colony.Preferably, cultivate these NT cells until at least about 2-400 cell, more preferably about 4-128 cell, and most preferably at least about 50 cells.Cultivation under appropriate condition, that is, and about 38.5 ℃ and 5%CO 2Carry out, be to optimize growth, typically every approximately 2-5 days, preferred per approximately 3 days replacing culture medium.
The method that is used for the processing of embryo transfer and receptor among the present invention is the used standard method of embryo transfer industry.Transplant synchronously for of the present invention successfully be important, promptly the oestrous cycle of NT embryo's period and recipient female animal is synchronous.Advantage and how supporting is estimated by uncle to see Sieolel, G.E.Jr. (" the evaluation comment of cattle embryo transplantation method " (1981) L. Mastroianni in external fertilization and the fetal development, Jr and J.D.Biggers compile, Plenum publishing house, New York, NY, 323 pages), its content is incorporated herein by reference.
By the present invention, be used for having of one's own somatic karyon cloning efficiency may with use identical from the karyon of fetal cell.For example, use from cow fetus and the stacked mulberry of adult cells whose development embryonic stage identical with blastocyst stage embryo's efficient.
The present invention also can be used for cloning genetic engineering or transgenic pig.As explained above, to have advantage be because transgenic method can be simplified by the cell source of differentiation that can clonal propagation is operated in the present invention.Especially, the required DNA sequence that has insertion, removal or modification as the noble cells of donor nuclei.Then cell those hereditary changes, differentiation is used for the nuclear transplantation of enucleation oocyte.
Any method that becomes known for inserting, remove or modify the target DNA sequence of mammalian cell all can be used for changing the noble cells of desire as nuclear donor.These methods can be removed all or part DNA sequence, and DNA sequence can be allogenic.Included technology has homologous recombination, one or more sequences of one or more specific site insertions, removal or modifying DNA that can be in cellular genome.
Therefore the present invention can be used for providing and has a required genotypic adult pig.The increase that has the adult pig of definite prepotency or other required character is useful especially, comprises transgenic or genetic engineering animal, and chimaeric animals.Therefore, the present invention can produce other offspring of monotypism, also can produce the pig that meat production increases, has the character and the disease resistance of reproduction.And,, comprise cell of transgenic and/or chimeric fetus and cell, tissue and the organ transplantation that tissue can be used for treating the multiple disease that is associated with use CICM cell as described below from the NT fetus.Therefore, transgenic pig has the purposes as the model of xenotransplantation that comprises disease, cell and organ and production pharmaceutical protein.
For producing CICM cell and cell line, after the NT unit that obtains desirable amount, cell is removed in manual operations from the zone, uses then.This process is preferably undertaken by following steps: get the cell mass that contains NT unit, this NT unit example ground comprises at least about 50 cells, washs these cells, and with the cell plating on feeder layer, Kuo San fibroblast for example.Typically, the cell that is used to obtain stem cell or cell colony can obtain from the innermost part of the NT unit that 50 cell sizes are preferably arranged of cultivating at least.But, the NT unit of less or greater number cell or also can be used for obtaining ES cell and cell colony from the cell of the other parts of NT unit.Cell is maintained suitable growth medium, for example, replenish with in the feeder layer among the α-MEM of 10%FCS and 0.1mM beta-mercaptoethanol (Sigma) and L-glutaminate.In order to help growth replaceable culture medium when the needs, changed once in for example about every 2-3 days.
This cultural method causes the formation of CICM cell or cell line.Those skilled in the art can change the growth that condition of culture is beneficial to specific CICM cell when needing.And, can produce the CICM cell of genetic engineering or transgenic pig according to the present invention.That is, said method can be used for producing the NT unit that has introduced one or more required DNA sequence, perhaps produces all or part of NT unit of having removed or having modified of wherein one or more endogenous DNA sequence.Those genetic engineering or transgenic NT unit can be used for producing the cell that genetic engineering or transgenic CICM cell comprise the people then.
The CICM cell and the cell that obtain, preferred people's CICM cell and cell line have in many treatments and the purposes in the diagnosis.Its specifically, these CICM cells can be used for cellular transplantation therapy.People CICM cell can be used for the treatment of numerous disease.People NT unit itself also can be used for treatment of diseases.
Consider this point, the embryo of known mice does (ES) cell can be divided into almost any cell type, for example hematopoietic stem cell.Therefore, the CICM cell of the pig that produces according to the present invention should have similar differentiation capability.CICM cell of the present invention can break up through inducing, to obtain required cell type according to known method.For example, by these cells of cultivation in division culture medium and under the confession condition of cell differentiation, but the pig CICM cell differentiation of induction experiment becomes hematopoietic stem cell, neurocyte, muscle cell, myocardial cell, hepatocyte, chondrocyte, epithelial cell, urethra cell, neurocyte etc.Cause the culture medium and the method for CICM cell differentiation known in the art, be called suitable condition of culture.
For example, Palacios etc. report to have taught among the 92:7530-7537 (1995) by stem cell being carried out following inducing in institute of NAS and produce hematopoietic stem cell from embryonic lineages, this abductive approach comprises: at first the aggregation of these cells is cultivated in the suspension culture base that lacks tretinoin, and then be incubated in the same medium that contains tretinoin, again cell aggregation is transferred on the substrate that makes cell attachment.
And, Pedersen, J.Reprod.Fertil.Dev., 6:543-552 (1994) is the survey article with reference to a large amount of articles, and the article of institute's reference discloses the method that cell type that embryonic stem cell produces multiple differentiation in vitro differentiation comprises hematopoietic stem cell, muscle, cardiac muscle, neurocyte etc.
And Bain etc. made progress in biology, had taught among the 168:342-357 (1995) to make embryonic stem cell produce the method for the neurocyte with neuron behavior in vitro differentiation.These lists of references are examples of the method that obtains noble cells from embryo or stem cell reported.The particularly wherein disclosed content that relates to the method that makes the embryonic stem cell differentiation of these lists of references is here quoted as a reference fully.
Therefore, use known method and culture medium, those skilled in the art can cultivate the CICM cell of this theme, comprise genetic engineering or genetically modified CICM cell, to obtain required differentiated cell types, and for example neurocyte, muscle cell, hematopoietic cell etc.
The CICM cell of this theme can be used for obtaining any required differentiated cell types.The therapeutic use of these noble cellss is unprecedented.For example, hematopoietic stem cell can be used in the therapeutic treatment that needs bone marrow transplantation.The method can be used to treat multiple disease, for example terminal cancer such as ovarian cancer and leukemia, and immune disease of mediation such as acquired immune deficiency syndrome (AIDS).Hematopoietic stem cell is obtainable, for example can be by ripe somatic cell such as epithelial cell or lymphocyte and non-nucleus egg mother cell fusion with cancer or acquired immune deficiency syndrome (AIDS) patient, obtain aforesaid CICM cell, and being beneficial to this cell of cultivation under the condition of differentiation, until obtaining hematopoietic stem cell.Such hematopoietic stem cell can be used in the treatment of diseases that comprises cancer and acquired immune deficiency syndrome (AIDS).
Optionally, can merge with non-nucleus egg mother cell, therefrom obtain people's CICM cell from the patient's of nervous disorder adult body cell, and with these cell culture under differentiation condition to produce neuronal cell line.Comprise for example parkinson disease, Alzheimer, ALS and cerebral palsy etc. by the specified disease of transplanting these human nerve cell treatments.In Parkinsonian special case, shown that the fetal brain neurocyte of transplanting normally is connected with on every side cell and produces dopamine.This can reverse symptoms of Parkinson's disease for a long time.
Remarkable advantage of the present invention has provided the main unlimited supply of the cell that waits gene or isogenic people that is suitable for transplanting.Therefore, it will eliminate the major issue relevant with existing implantation method promptly, because the host is to graft or transplant the repulsion of the contingent transplanted tissue that the repulsion to the host causes.Traditionally, by resisting-repel medicine such as cyclosporin to prevent or reducing repulsion.Yet these medicines have significant adverse side effect such as immunosuppressant, carcinogenecity and very expensive.The present invention should eliminate, and perhaps significantly reduces the requirement to anti-rejection drugs at least.
Other available disease and disease of gene cell treatment of waiting comprises that for example spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic disease are hypercholesterolemia, heart disease, cartilage displacement, burn, ulcer of foot, gastrointestinal disease, angiopathy, kidney disease, urethral disease and aged relevant disease and disease.
The method can be used for replacing dcc gene, as, the immune system gene of defective, cystic fibrosis gene perhaps having imported to the gene that protein useful on making a study of subjects such as somatomedin, lymphokine, cytokine, enzyme etc. are expressed.For example, the DNA sequence of coding brain-derived growth factor can be imported people CICM cell, these cell differentiation neuroblasts, and with these nerve cells transplantations to the parkinson patient to stop the forfeiture of neurocyte in the lysis.
In the past, be different with BDNF cells transfected type primary cell line with fixed cell system, not the cell with regard to right and wrong nerve (sarcoplast and fibroblast) source in neural source.For example, adopted retroviral vector BDNF gene transfection astrocyte, and these cell transplantations have been entered (Yoshimoto etc., brain research, 691:25-36, (1995)) in the Parkinsonian rat model.
Transplant after 32 days, the treatment of this ex vivo has reduced the parkinson symptom of rat up to 45%.Also have, Tyrosine Hydroxylase Gene is inserted astrocyte similar result (Lundberg etc., developmental neurobiology, 139:39-53 (1996) and the reference of wherein introducing).
Yet this is from from intravital system problem being arranged.Particularly, at present the used retroviral vector gene reducing in vivo and shift be transient expression (Mulligan, summary, science, 260:26-932 (1993)).And, used primary cell in these researchs, vital stage that astrocyte is limited and duplicating slowly.These characteristics have influenced the speed of transfection unfriendly and have hindered the screening of the cell of stable transfection.And the primary cell of breeding gene targeting used in a large amount of homologous recombination techniques almost is impossible.On the contrary, the cell of applications exploiting differentiation and the mammiferous clone of CICM cell should eliminate relevant with the retrovirus system.
The DNA sequence that can introduce the CICM cell of this theme comprises, for example, the DNA sequence of the enzyme of those coding schedule skin growth factors, basic fibroblast growth factor, the deutero-neurotrophic somatomedin of neuroglia, insulinoid somatomedin (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine (interleukin, interferon, colony stimulating factor, tumor necrosis factor (α and β) etc.), treatment usefulness etc.
The application of people's NT unit and CICM cell, the present invention also comprises the application of cell inhuman in people's the disease treatment in cell, tissue and organ transplantation.Therefore, CICM cell, NT fetus and NT that plants arbitrarily and chimeric offspring (genetically modified or not genetically modified) can be used for the treatment that cell, tissue and organ transplantation are proper human diseases.In general, can be used between (from body, isogenic or allograft) mutually of the same race or kind and the kind (xenotransplantation) according to CICM cell of the present invention, fetus and offspring.For example, the brain cell from cattle NT fetus can be used for treating parkinson disease.
And the CICM cell of this theme also can be used as differentiation, particularly regulates the external model of the research of relevant gene with early development.And the cell, tissue and the organ that go out with the CICM cell differentiation of this theme also can be used in the drug research.
And the CICM cell of this theme can be used as the nuclear donor that produces other CICM cell and cell colony.
In order more clearly to describe the present invention, the following examples are provided.Separating of 1 N of embodiment and pig embryo and the fibroblastic primary culture of adult cattle
The fibroblastic primary culture of cattle and pig obtains from fetus (cattle became pregnant 45 days and pig fetus 35 days).Aseptic removal head, liver, heart and digestive tract, chopping fetus and at the trypsin EDTA of pre-incubation solution (0.05% trypsin/0.02%EDTA; GIBCO, Grand Island cultivated 30 minutes in 37 ℃ in NY).Be inoculated in fibroblast in tissue culture's ware and containing 10% hyclone (FCS) (Hyclone, Logen, UT), α-MEM culture medium (BioWhittaker, Walkersville, MD) the middle cultivation of penicillin (100IU/ml) and streptomycin (50 μ l/ml).Fibroblast is containing 5%CO under 37 ℃ of wet condition 2Air in cultivate and keep.Cell reaches that routine goes down to posterity when being paved with.
Adult fibroblast separates with skin from the lung in milch cow (about five years old age).The chopping lung tissue in 10 ℃ at trypsin EDTA solution (0.05% trypsin/0.02%EDTA; GIBCO, GrandIsland, NY) middle overnight incubation.Second day, the tissue and the cell of any disengaging in 37 ℃ at the trypsin EDTA of pre-incubation solution (0.05% trypsin/0.02%EDTA; GIBCO, GrandIsland cultivated 1 hour in NY) and carries out three continuous washing and trypsin is cultivated (1 hour).Be inoculated in fibroblast in tissue culture's ware and at the α that augments-MEM culture medium (BioWhittaker, Walkersville cultivates in MDC) approximately from the blastodisc after date and grows a period of time (12-15 days to 10 years old-15 years old animal of cattle after fertilization) to the animal manhood.The method also can be used for comprising that from other mammals mice is separated into fibrocyte.Marker gene (external allogeneic dna sequence DNA) is imported embryo and adult fibroblast.
Embryo (cattle and pig) and adult (cattle) fibroblast are carried out following electroporation operation.The standard micro-injection method also can be used for allogeneic dna sequence DNA is imported fibroblast, yet, because operation is easier, so adopt the electroporation operation in the present embodiment.
To contain fibroblastic culture plate of breeding and place trypsin EDTA solution (0.05% trypsin/0.02/EDTA; GIBCO, Grand Island, NY) the middle cultivation becomes the individual cells suspension until cell.The rotation sedimentation cell also makes every milliliter to contain 5 * 10 with phosphate buffered saline (PBS) (PBS) suspension cell again under 500xg 6Individual cell.
The reporter gene construct comprises cytomegalovirus promoter and beta galactosidase, neomycin phosphotransferase fusion gene (β-GEO).With reporter gene and final concentration is that the cell of 50 μ g/ml is added in the electroporation cell.After the electroporation pulse, with fibroblast shift get back to growth medium (contain 10% hyclone (FCS) (Hyclone, Logen, UT), the α-MEM culture medium (BioWhittaker of penicillin (100IU/ml), streptomycin (50 μ l/ml), Walkersville, MFD)) in.
After the electroporation, screen adherent fibroblast and be used for the stable integration of reporter gene.G418 (400 μ g/ml) is added 15 days (scopes: 3 days life end of term up to cultured cells) of effect in the growth medium.This medicine kills all cells that do not have β-GEO gene, and reason is that they are not expressed neo resistance base and exist.In latter stage during this period of time, the colony of stable transgenic cell appears.Breeding independently between each colony is mutual.With X-gal with transgenic fibroblast dyeing with the expression of observing beta galactosidase and turn out to be the positive be used to use β-GEO gene pcr amplification integration and on agarose gel, run band.In the nuclear transplantation method, use the transgenic fibroblast to create CICM cell line and transgenic fetus
To derive from a cell line (CL-1) of a colony behind the cattle embryo fibroblast as the donor karyon in nuclear transplantation (NT) method.The conventional method of NT has been described above.
The slaughterhouse oocyte is at maturation in vitro.The micropipet enucleation that these oocytes are peelled off upright cell and about 18 to 20 hours (hpm) uses inclination after maturation.Add Hoechst 33342 (3 μ g/ml in the TL-HEPES culture medium; Sigma) confirm enucleation in.Then single donorcells (fibroblast) is placed the ovum week crack of receptor oocyte.Answer the electricity consumption integration technology that bovine oocyte Cytoplasm and donor nuclei (NT unit) are merged.Being full of the once fusion pulse that acts on NT unit in the gap cell that merges culture medium at 500 μ m is 120V 15 seconds.This occurs in ripe back 24hpm.Place the CRlaa culture medium up to 26 to 27hpm NT unit.
The conventional method that is used for manually activating oocyte has been described above.The activation of NT unit is 26 and begin during 27hpm.In brief, NT unit is exposed to ionomycin (5 μ M among the TL-HEPES that contains 1mg/ml BSA; CalBiochem, La Jolla, CA) 4 minutes and washed 5 minutes with the TL-HEPES that contains 30mg/ml BSA.In the ionomycin processing procedure, also NT unit is placed 2mM DMAP (Sigma).After the flushing, NT unit is transferred in the CRlaa culture medium microdroplet that contains 2mMDMAP (Sigma) and at 38.5 ℃, 5%CO 2Under cultivated 4 to 5 hours.Wash the embryo and place the CRlaa culture medium of four well culture plates that contain mouse embryo fibroblasts fusion feeder layer to add 10%FCS and 6mg/ml BSA then.At 38.5 ℃ and 5%CO 2Cultivate NT unit under the condition more than 3 days.Changed a subculture in per three days up to activating back 5 to 8 days.This moment, blastocyte phase NT embryo can be used for producing transgenic CICM (inner cell mass of cultivation) cell line or fetus.The inner cell mass of separable these NT units also is planted on the feeder layer.And, NT unit is transferred in the recipient female animal.In the back interruption of pregnancy in 35-48 days of becoming pregnant.This produces the transgenic fetus that has 7 clones of β-GEO gene in the tissue of all inspections.Detecting in 7 fetuses by ultrasonic observation has 6 to have normal heart beating.And the tissue slice of fetus does not show significantly unusual.The method generally helps gene targeting CICM cell line and fetus.
Following table has been summed up these result of experiment.
The donorcells type ????n The spilting of an egg (%) Blastocyte (%) CICM system *?????(％) The transgenic fetus (%) that regains Continue gestation after 40 days
CL-1 cattle tire fibroblast (bGEO) ????412 ?220(53％) ??40(10％) ????22(55％) ????N/A ????N/A
CL-1 cattle tire fibroblast (bGEO) ????3625 ?2127(59％) ??46(9％) ????N/A 7 fetuses + ????8 ≠
CICM cell line from the CL-1NT embryo ????709 ??5(0.7％) ????N/A ????0 ????0
The cattle fibroblast grows up ????215 ?119(55％) ??20(9％) ????N/A ????N/A ????N/A
* 19 systems are β-GEO positives, 2 feminine genders and a death before PCR detects.In 35 days the growth of+gestation, a foetal death, another growth is slightly slow.5 fetuses that gestation regained in 38-45 days are normal.All fetuses turn out to be genetically modified.≠ first offspring is born in October, 1997.Embodiment 2 derives from the chimeric fetus and the offspring of transgenic CICM cell.Transgenic CICM cell line is at first from transgenic NT unit (noble cells).
The CICM cell line that derives from transgenic NT embryo (the CL-1 cell transfer is entered non-nucleus egg mother cell) is used for producing chimeric embryo and fetus.With 1-5mg/ml pronase or 0.05% trypsin/EDTA in conjunction with the depolymerization of mechanical depolymerization method render transgenic CICM cell colony, so that produce 5 or cell mass still less.Make trypsin or the active inactivation of pronase by repeatedly wash cell with 30～100% hyclones.The cell of these depolymerization is placed the micrurgy plate that contains the TL-HEPES culture medium.Also the embryo who is fertilized is placed these plates and uses the micrurgy instrument and produce chimeric embryo.8 to 10 transgenic CICM injection cells are entered among the fertilization embryo of 8-16 cell stage.These embryos of In vitro culture enter in the receptor to blastocyst stage and transplanting subsequently.
6 the non-surgery of blastocyst stage chimeric embryo ground are transplanted and are entered in two recipient female animal altogether.After conceived 5 weeks, regain 3 fetuses.Several tissues of these three fetuses comprise that the screening of the sexual cell (expression germline mosaic) of gonad is by the Southern blot hybridization to segmental pcr amplification of beta galactosidase and amplified production.In these three fetuses, two is from the transgenic CICM cell contribution positive.These two fetuses all have transgenic CICMcontribution gonad.
Allow 10 chimeric embryos to foot day, wherein give birth to the offspring for 7.Make the ear incisura and from each calf DNA isolation.Pcr amplification one, one of them turns out to be the chimeric offspring of transgenic.Transgenic NT embryo is from transgenic CICM cell line.Transgenic CICM cell line is at first from transgenic NT unit (cell of differentiation)
Identical transgenic CICM cell line is used to produce the NT embryo.Except what be used as the fusion of donorcells and non-nucleus egg mother cell is CICM cell rather than the fibroblast, and having used embodiment 1 is described NT method.With 1-5mg/ml pronase or 0.05% trypsin/EDTA in conjunction with the depolymerization method render transgenic CICM cell colony depolymerization of machinery so that produce 5 or still less cell mass.It enters cell transplantation before the enucleation oocyte, and the repeatedly flushing of carrying out the 30-100% hyclone by pair cell makes trypsin or the active inactivation of pronase.Reported result's (the 3rd group) in the table 1.Five blastocyst stage embryos have been produced.

Claims (87)

1. the method for a cloning mammal comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation.
(ii) activate the nuclear transplantation unit that obtains; And
(iii) the NT unit with described cultivation is implanted into host mammal so that NT unit develops into fetus.
2. method according to claim 1 further comprises making fetal development become the offspring.
3. method according to claim 1 is wherein inserted in the mammalian cell of described differentiation or nucleus, is removed or modifies target DNA, thereby causes the generation of the NT unit of hereditary change.
4. method according to claim 3 further comprises making fetal development become the offspring.
5. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are from the mesoderm pedigree.
6. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are from the ectoderm pedigree.
7. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are from the entoderm pedigree.
8. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are fibroblast or nucleus.
9. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are from ungulate.
10. method according to claim 9, wherein ungulate is selected from cattle, sheep, pig, horse, goat and Babalus bubalis L..
11. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are into somatic cell or nucleus.
12. method according to claim 1, wherein Fen Hua mammalian cell or nucleus are embryo or fetal cell or nucleus.
13. method according to claim 1, wherein non-nucleus egg mother cell is ripe before enucleation.
14. method according to claim 1, wherein the nuclear transplantation unit of Rong Heing is by being exposed to ionomycin and the 6-dimethylamino-purine activates.
15. method according to claim 3 wherein adopts microinjection to insert allogeneic dna sequence DNA.
16., wherein adopt electroporation to insert allogeneic dna sequence DNA according to the method for claim 3.
17. the fetus that obtains according to the method for claim 1.
18. the offspring who obtains according to the method for claim 2.
19. offspring's according to claim 18 filial generation.
20. the transgenic fetus that obtains according to the method for claim 3.
21. the transgenic progeny that obtains according to the method for claim 4.
22. offspring's according to claim 21 filial generation.
23. method according to claim 1 comprises that further the NT unit with the clone combines to produce chimeric embryo with the fertilization embryo.
24. method according to claim 23 further comprises making fetal development become the offspring.
25. the fetus that obtains according to the method for claim 23.
26. the offspring who obtains according to the method for claim 24.
27. mammiferous filial generation according to claim 26.
28. method according to claim 1 is wherein cultivated described activatory nuclear transplantation unit until surpassing the 2-cell development stage.
29. produce the method for CICM cell line, comprising:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(ii) cultivate the cell that obtains from the NT unit of described cultivation to obtain CICM cell line.
30. method according to claim 29 is wherein cultivated described activatory nuclear transplantation unit until surpassing the 2-cell development stage.
31. the CICM cell line that obtains according to the method for claim 29.
32. method according to claim 29 is wherein inserted in the mammalian cell of described differentiation or nucleus, is removed or modifies target DNA, thereby causes the generation of the NT unit of hereditary change.
33. transgenic CICM cell line according to claim 32 acquisition.
34. method according to claim 29 is wherein induced the CICM cell line differentiation that obtains.
35. the noble cells that obtains according to the method for claim 34.
36. the people's noble cells that obtains according to the method for claim 34.
37. a Therapeutic Method comprises that the patient who needs cellular transplantation therapy is with the gene noble cells that waits according to claim 36.
38. the method for claim 37 is wherein carried out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer.
39. a Therapeutic Method comprises that the patient who needs cellular transplantation therapy is with allogene noble cells according to claim 35.
40. according to the described method of claim 39, wherein the allogene noble cells is the cattle cell.
41. the method for claim 39 is wherein carried out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer.
42. the method for claim 37, wherein Fen Hua people's cell is hematopoietic cell or neurocyte.
43. the method for claim 37, wherein therapy is to be used for the treatment of parkinson disease and noble cells is a neurocyte.
44. the method for claim 37, wherein therapy is to be used for the treatment of cancer and noble cells is a hematopoietic cell.
45. a Therapeutic Method comprises the heterogenote of patient to obtain from fetus according to claim 17 that needs cellular transplantation therapy.
46. according to the described method of claim 45, wherein heterogenote is the cattle cell.
47., wherein carry out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer according to the described method of claim 45.
48., wherein carry out described cell transplantation therapy with the treatment parkinson disease according to the described method of claim 46.
49. a Therapeutic Method comprises the heterogenote of patient to obtain from offspring according to claim 18 that needs cellular transplantation therapy.
50. according to the described method of claim 49, wherein heterogenote is the cattle cell.
51. the method for claim 49 is wherein carried out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, a kind of farm tools urine disease, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer.
52. a Therapeutic Method comprises the allogene transgenic cell of patient to obtain from transgenic fetus according to claim 20 that needs cellular transplantation therapy.
53. according to the described method of claim 52, wherein the allogene transgenic cell is the cattle cell.
54. the method for claim 52 is wherein carried out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, a kind of farm tools urine disease, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer.
55. the method for claim 53 is wherein carried out described cell transplantation therapy with the treatment parkinson disease.
56. a Therapeutic Method comprises the allogene transgenic cell of patient to obtain from transgenic filial generation according to claim 21 that needs cellular transplantation therapy.
57. according to the described method of claim 56, wherein the allogene transgenic cell is the cattle cell.
58. the method for claim 56 is wherein carried out described cellular transplantation therapy to treat following disease or disease: parkinson disease, Huntington Chorea, Alzheimer, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic disease, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, angiopathy, urethral disease, AIDS and cancer.
59. method according to claim 29 comprises that further the NT unit with the clone combines to produce chimera with the fertilization embryo.
60., further comprise making chimeric CICM cell line develop into chimeric embryo according to the described method of claim 59.
61. chimeric embryo according to claim 60 acquisition.
62., further comprise making chimeric embryo develop into chimeric fetus according to the described method of claim 60.
63. chimeric fetus according to claim 62 acquisition.
64., further comprise making chimeric fetal development become chimeric offspring according to the described method of claim 62.
65. chimeric offspring according to claim 64 acquisition.
66. according to the described method of claim 59, wherein in the mammalian cell of described differentiation or nucleus, insert, remove or modify target DNA, cause the generation of the NT unit of hereditary change thus.
67., further comprise making chimeric CICM cell development become chimeric embryo according to the described method of claim 66.
68. chimeric embryo according to claim 67 acquisition.
69., further comprise making chimeric embryo develop into chimeric fetus according to the described method of claim 67.
70. chimeric fetus according to claim 69 acquisition.
71., comprise that further chimeric fetal development becomes chimeric offspring according to the described method of claim 69.
72. chimeric offspring according to claim 71 acquisition.
73. the method for a cloning mammal comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required CICM cell non-serum starved, differentiation or the cell or the nucleus enucleation mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(iii) the NT unit with described cultivation is transferred in the host mammal so that NT unit develops into fetus.
74., wherein cultivate described activatory nuclear transplantation unit until surpassing the 2-cell development stage according to the described method of claim 73.
75., further comprise making fetal development become the offspring according to the described method of claim 73.
76. the fetus that obtains according to the method for claim 73.
77. the offspring who obtains according to the method for claim 75.
78. be used for the heteroplastic organ of organ from what offspring according to claim 18 obtained.
79. be used for the heteroplastic organ of organ from what offspring according to claim 21 obtained.
80. be used for the heteroplastic organ of organ from what offspring according to claim 26 obtained.
81. be used for the heteroplastic organ of organ from what obtain according to the described offspring of claim 72.
82. be used for the heteroplastic organ of organ from what obtain according to the described offspring of claim 77.
83. method according to claim 13, wherein non-nucleus egg mother cell is at maturation in vitro.
84. method according to claim 1, wherein activation occurs in after the formation of NT unit.
85. 4 described methods according to Claim 8, wherein 4 hours or more late activation the after NT unit forms.
86. method according to claim 1, wherein with NT unit in uncertain culture medium with accessory cell in external co-cultivation.
87. one kind prepares the proteic method of pharmaceutically active, comprises the pharmaceutically active albumen that separation is expressed by transgenic progeny according to claim 21.
CN98807691A 1997-07-03 1998-06-24 Cloning using donor nuclei from non-serum starved, differentiated cells Pending CN1265599A (en)

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