CN1424870A - Nuclear transfer with selected donor cells - Google Patents

Abstract

The present invention provides a method of nuclear transfer by selecting and segregating G1 cells from a donor cell population. This method is advantageous over the prior art as it provides certainty as to the stage of the cell cycle which the donor nuclei are in and allows for the production of cloned transgenic or non-transgenic embryonic cells, reconstituted embryos and whole animals for agricultural, pharmaceutical, nutraceutical and biomedical applications.

Description

Implement nucleus transplantation with the donorcells of selecting

The present invention relates to a kind of new method of nucleus transplantation, in particular, produce mammal embryo, fetus and offspring, comprise in genetic engineering or transgene mammal embryo, fetus and offspring's the clone technology but never repel to be used in.

Background of invention

Donorcells nuclear and the time phase of recipient cell kytoplasm cell cycle of living in were the key factors that success is grown after the decision nucleus transplantation when embryo rebuild.The particular combinations of two kinds " cells " is to guarantee that for the first time cells,primordial week after date obtains a cover dliploid chromatin and cell nucleus rearranged and growth chance subsequently is increased to necessary to greatest extent.When the nuclear of the donorcells during the cell division merges with stoning metaphase arrest egg mother cell (being called cytoplasm or recipient cell), nuclear membrane disintegration (NEBD) and donor chromatin take place immediately suffer premature chromosome condensation (PCC) (Barnes etc., 1993).These effects are to be called maturation-promoting factor by one (MPF is also referred to as short reduction division or mitogenic factor, reference, summary, Campell etc., cytoplasm activity inducement 1996a).In oocyte maturation, the activity of MPF is the highest in mid-term, and descends rapidly behind fertilization or artificial activation.So two types cytoplasm can be used for rebuilding; II in mid-term (M II) cytoplasm of use un-activation or activation is corresponding to be the higher or lower successively cytoplasm of MPF content.

Helping to recognize in the mammalian nuclear transfer, using in the early stage research work of cell cycle harmony to the importance of the potentiality of development that keeps the chromosome complete sum and cause thus from such as the not differentiation blastomere of species preimplantation embryo such as rabbit, sheep and ox (promptly not specialization embryonic cell).These researchs disclose the time span that donorcells examines the stage of cell cycle of living in and be exposed to cytoplasm MPF observed PCC degree are had remarkable influence.There is typical pulverization profile in the S phase nuclei dyeing chromaticness that is exposed among the MPF, and chromosome research has shown the rate occurred frequently of deformity.(Collas etc., 1992b).On the contrary, the nuclear chromatin condensation that is in G1 or G2 phase form the chromosome of the elongation that has sub-thread or bifilar chromatid successively (Collas etc., 1992b).To rebuild after the embryo frees from metaphase arrest and activate the spread effect of growth suitable, nuclear membrane is around the reorganization of donor chromatin, no matter cell cycle phase of living in before it subsequently, it is synthetic that donor chromatin carries out DNA.So, the donorcells that is in the G1 phase authorizes that to have moved the DNA compatible with normal development synthetic, the cell nucleus that is in G2 phase or S phase then completely or partially duplicates the DNA that has duplicated again, so consequently, during to cells,primordial end cycle for the first time, the dna content of two daughter cells is incorrect, causes unusual early embryonic development.

On the contrary, these studies show that after cytoplasm activates in early days through a sufficiently long interval, after the MPF disappearance, in cytoplasm, the stage control dna replication dna of (so PCC does not take place yet) and donorcells nuclear according to its cell cycle of living in when transplanting just do not take place in NEBD the blastomere nuclear transplantation.So the cell nucleus of G1 phase or S phase starts respectively or continues and duplicates, it is synthetic that an other DNA then can not be induced or enter to the cell nucleus of G2 phase.This cytoplasm that activates in advance is named as " crf receptor " (Compbell etc., 1994), and it had in any stage of cell cycle coordinates the ability that donorcells is grown.This point is to clone's preimplantation embryo particular importance, and great majority do not break up the blastomere cell nucleus and are in S phase (80-90%, Barnes etc., 1993 at any time in preimplantation embryo; Campbell etc., 1994), so the most suitable being transplanted to contains in the lower cytoplasm of MPF.

After the nucleus transplantation, the factor that (perhaps external cause induce co-factor) has the chromatin Structure reinvented and suitably rearrange donorcells nuclear gene expression-form is depended in the egg mother cell cytoplasm in normal development.Mature cell matter comprises rna transcription thing and the protein that the normal activationary time of genome is arrived in the cleavage stage embryonic development of instructing normal fertilization, and the normal activationary time of genome is meant that blastomere nuclei begins the synthetic RNA that controls oneself and and guides embryonic development.So it is synthetic to examine the RNA that must stop them after reconstruction from the donorcells of embryo who crosses the normal activationary time point of genome or cell type, and keep inactivated state to take place up to the new parent embryonic gene group conversion of rearranging.After the transplanting, donorcells nuclear is forced to the zygotic state of rearranging, and with normal embryo development required correct level takes place subsequently, and suitable space-time approach activates suitable gene.Realize that the mechanism that this cell nucleus is rearranged is not fully aware of at present.

In view of the interest of carrying out nucleus transplantation with the cell of cultivate preserving recently, rearrange and the cell cycle coordinates to have become important topic.Cultured cells is broken up (promptly having the cell function of specialization more) more than the cell that is used for early stage research with embryo's blastomere clone.These cells can separate from embryo, fetus or animal adult.Because can obtain the cell of larger amt, so the production of transgenic animal the efficient nuclear transfer technology of use noble cells culture may be realized the genotype that extensive propagation is wished and will speed up the cultured cell genetic manipulation in domestic animal after.

Early stage (Campbell etc. with active growth, not synchronized sheep blastocyte (cell cycle is unclear) culture, 1995) but be not that (Campbell etc. 1996b) have in fact produced the term lamb with the early stage research that preceding active cell matter merges for the passage cell in later stage.Spend the cell that removes serum after 5 days, remain static (promptly, cell through this processing is considered to withdraw from normal cell division cycle, enter so-called G0 state) to its before, the follow-up study of the cytoplasm fusion that activates with similar overall efficiency thereafter or simultaneously produced lamb alive (Campbell etc., 1996b).These authors (Campbell etc., 1996b; Schnieke etc., 1997; Wilmut etc., 1997; Patent WO97/07669) proposed to use to withdraw from the normal cell division cycle and be synchronized to cell static or the G0 state and help cell nucleus to rearrange and produce the importance of cloning animal from noble cells.The static synchronized method of preferred realization cell (to lack proliferative cell cell nuclear antigen (PCNA), this kind situation shows does not have cell to be in the S phase) that is proposed by above-mentioned author is the serum starvation of suitable time.Yet, recently, be subjected to query about the cell proportion of the actual G0 of being in state in the serum starvation cell.Use two parameter flow cytometries to measure cell DNA and protein content simultaneously (in order in dliploid colony, to distinguish G0 and G1 phase cell, resting stage, cell contained still less RNA and albumen), Boquest etc. (1999) have studied the cell cycle feature of the porcine fetus fibroblasts of cultivating.Though they confirm that the serum starvation passed through 5 days handles, in fact be lower than 50% cell and be in the G0 state according to their definition.By selecting " little " cell in the cell mass, G0 cell share is increased to 72% (Boquest etc., 1999) in the serum starvation culture.

As the alternative method of using cell resting stage, Cibelli and colleagues (1998; With patent specification WO 99/01163) reported that free growing ox cell culture that uses non-serum starvation and II in mid-term (M II) cytoplasm that uses ionomycin and 6-dimethylamino-purine (6-DMAP) to activate subsequently merge.But these bulletins are not illustrated donorcells cell cycle of living in when nucleus transplantation that these methods of use finally produce cloning calf embryo.

Similarly, other report cloning calf (Vignon etc., 1998,1999; Zakhartchenko etc., 1999) and mouse (Wakayama and Yangimachi, 1999) from the free auxocyte culture of non-serum starvation, produce.But as before, the stage that produces donorcells cell cycle of living in when carrying out nucleus transplantation of cloning offspring be can not determine.

So, above the research carried not have which that confirms the cell cycle exactly or which stage to cause rebuilding among the embryo the final ratio that survives the offspring of growing so low in a stage.

Above-mentioned discussion emphasizes, up to now, about few generally speaking pitiful of the donorcells the cultivated description of cell cycle of living in when the nucleus transplantation, this just makes the present technique horizontal instability and is difficult for repeating.

So, expect to have a kind of nucleus transplantation method that donorcells is examined cell cycle phase of living in of accurately knowing.

One object of the present invention is exactly to push ahead so that realize this expectation or provide the choice of usefulness at least to the public.

Summary of the invention

According to first aspect, the invention provides a kind of method of nucleus transplantation, this method comprise from propagation or do not select the donorcells group of propagation and separate in G1 phase cell and the recipient cell from the G1 phase cell that separates like this nuclear transplantation to a stoning.The donorcells group can be in the one or more known or unknown stage of cell cycle.

This method provides the certainty about donorcells nuclear stage of cell cycle of living in when the embryo rebuilds, so be better than prior art.

The present invention considers to use cell cycle inhibitor that free growing cell is arrested in the moment of cell cycle, so that produce non-propagation synchronization cell group.In a single day preferably, the cell stunt stagnates in mitosis, and removes inhibitor, the cell that enters the G1 phase is used for nucleus transplantation.

So, aspect second, the invention provides a kind of nucleus transplantation method, this method comprises in the recipient cell with nuclear transplantation to a stoning of an isolated cells from the non-proliferating-cell population that is synchronized with the cell cycle G1 phase.

Preferably, described G1 phase cell is from free proliferating cells group or single separation from be in the early stage non-propagation synchronization cell group of G1.

Selectively be that non-proliferating-cell population can comprise senile cell.

The G1 phase donorcells that separates is in animal body, perhaps, more preferably, separates from vitro cell culture.The cell that is fit to can be from the embryo, fetus, and growing animal is up to full ripe adults.In fact, any normal diploid cell of caryogram or senile cell that possesses ability of cell proliferation may be used among the present invention.Cell can be undifferentiated state or cell differentiation in various degree, as long as they can be entered cell cycle and propagation by stimulation.The cell of resting stage can stimulate with suitable condition of culture (for example adding serum or special growth factor) and enters the cell cycle and the G1 after mitosis is used for nucleus transplantation in early days.It is more effective than other type that some cell types can be proved to be, but fibroblast of adult and fetus and adult follicle cell have been found satisfactory.For the present invention is described, be presented in the result of implementation that (embodiment 1-7) uses the fetal fibroblast of two kinds of follicle cell systems, four kinds of skin fibroblast lines and two kinds of genetic modifications to be in the ox below.

Preferably, recipient cell comprises the enucleation oocyte from the species consistent with donorcells nuclear source.Enucleation oocyte can have higher or lower MPF activity when the embryo rebuilds, because two states is to hold with the G1 phase donorcells nuclear phase with 2C content DNA.

Selectively be that recipient cell can comprise the stem cell of a stoning or the stoning stem cell that merges.Preferably, these stem cells are embryonic stem cells.The embryonic stem cell itself that is used as recipient cell in nucleus transplantation can separate from embryo who is growing or the culturing stem cells of having set up system.In this case, the nucleus transplantation done of the donorcells of the G1 phase cell of selecting with method of the present invention nuclear can be used for the purpose of " therapeutic cloning ".

According to the 3rd aspect, the invention provides G1 phase donorcells nuclear by separating, preferably G1 is early stage, is transplanted in the recipient cell of stoning and produces the method for cloning animal embryo.

Method of the present invention can be used to produce any animal embryo, about species comprise birds, amphibian animal, fish and mammality.Preferably, relevant animal embryo is a mammal embryo, include but not limited to, and primate comprises people, rodent, rabbit, cat, dog, horse, pig and most preferably, ungulate such as ox, sheep, deer and goat.

Preferably use the cloned animal embryo that cell nucleus obtained of the method genetic modification known to the present technique field to have the genetic character of expectation.

According to the 4th aspect, the invention provides the recombinant animal embryo of a kind of usefulness method preparation of the present invention, comprise reorganization transgenic animal embryo.So the embryo who forms then again time cloning so that further increase embryo's quantity or carry out that continuous kernel is transplanted so that further help nuclear to rearrange and/or potentiality of development.

According to the 5th aspect, the invention provides a kind of non-human animal's of clone method, this method comprises that (1) produces the cloning animal embryo according to the method that invention recited above provides; (2) with known method by animal from the embryonic development to the childbirth; And (3) are with conventional method or according to the inventive method clone's the random breeding animal that forms like this.

Method of the present invention can be used to produce the non-human animal, and relevant species comprise birds, amphibian animal, fish and mammality.Preferably, said non-human animal is selected from: the primate beyond the people, rodent, rabbit, cat, dog, horse, pig and most preferably, ungulate such as ox, sheep, deer and dog.

According to the 6th aspect, the invention provides with the method in the above-described invention and prepare non-human cloning animal, and offspring and the descendant of this non-human cloning animal.

Method of the present invention can be used to produce the non-human animal, preferred mammal, and it has the expectation genetic character of using present technique field known method genetic modification donorcells nuclear to be obtained.The transgenic animal of Chan Shenging also constitute a part of the present invention in this way.

The present invention also can be used for producing and is applicable to, the embryo cell line of cell, tissue and organ transplant for example, embryonic stem cell line, fetus or offspring.

Correspondingly, in another embodiment, the invention provides the method that produces embryo cell line, this method comprise the steps: a) from the donorcells propagation group who does not know the cell cycle or select in the synchronized G1 phase cell mass separate G1 phase cell and with the nuclear transplantation of isolated cell in the stoning recipient cell; B) cultivate blastocyst stage; C) reclaim embryonic cell; And d) use method well-known in the art to set up external immortalized cell system.

In another embodiment, the method of producing animal embryonic stem cell is provided, this method comprise the steps: a) from the animal donor cell growth group of the different phase that is in cell cycle the unknown or select in the synchronized G1 phase culture separate G1 phase cell and with the nuclear transplantation of isolated cell in the stoning animal recipient cell; B) cultivate blastocyst stage; C) reclaim embryonic stem cell.

Preferably, the animal donor cell that uses in said method is the people source.Most preferably, animal donor of using in said method and recipient cell all are the people sources.

Most preferably, donorcells is adult or the fetal cell that is selected from the normal cell type of any caryogram, and recipient cell is to be selected from any cell type of gene expression of can rearranging, and comprises enucleation oocyte or embryonic stem cell.

The embryonic stem cell that produces with method of the present invention is a multipotential cell, can in cultivation, induce the purifying group who is differentiated to form human body cell specialization type with method well-known in the art, comprise nerve cell for example neuron, astroglia and oligodendroglia; Liver cell; Muscle cell is myogenous cells for example; Heart cell is the cardiac muscle cell for example; Hematopoietic cell; Pancreatic cell and other interested cells.

The human body cell of these specializations and organize then can specified disease or damaged cell can not self or the injury that can not effectively upgrade in be used for transplantation treatment.Wherein, people's donorcells is from the patient of this transplanting of needs, because the tissue of transplanting is identical in heredity with patient, the tissue of this transplanting just can not repelled by patient.

Alternately be, the cell of these differentiation and tissue can be used for the treatment of i or I, for example, multiple nervous system disorders (for example Parkinson's), diabetes, cardiopathy, amyotrophy, multiple hereditary disease, particular cancers (for example leukemia), spinal cord injury, burn and other miseries.

These methods are called " therapeutic cloning " in the present technique field.

Human embryo stem cell vitro differentiation becomes particular cell types also to help screening of medicaments and human medicine toxicologic study.

So, in yet another aspect, the invention provides the method for carrying out drug screening and drug toxicology experiment with the inventive method production vitro differentiation human embryo stem cell.

According to the another one aspect, the invention provides heteroplastic method, wherein cell, tissue and organ can separate from the non-human cloning animal of the method according to this invention production and their offspring, are used for the human diseases patient's of this treatment of needs transplanting.If comprised transgenosis in this cell, tissue or the organ, this cell, tissue or organ can be used for gene therapy, or regulate the immune response of patient to the tissue that contains foreign gene.

Description of drawings

With reference now to accompanying drawing, the present invention is described:

Survival rate in the complete pregnant process of cloning ox embryo that Fig. 1 represents to rebuild with G0 that is in the donorcells cycle or G1 phase follicle cell;

Survival rate in the complete pregnant process of cloning ox embryo that Fig. 2 represents to rebuild with G0 that is in the donorcells cycle or female adult SF of G1 phase;

Survival rate in the complete pregnant process of cloning ox embryo that Fig. 3 represents to rebuild with G0 that is in the donorcells cycle or female adult SF of G1 phase (3XTC cell);

Embryo survival in the complete pregnant process of cloning ox embryo that Fig. 4 represents to rebuild with G0 that is in the donorcells cycle or male adult SF of G1 phase (LJ801 cell);

Fig. 5 represents with the embryo survival in the complete pregnant process of cloning transgenic cow embryo that is in the G0 in donorcells cycle or G1 phase, rebuilds through the female fetal lung fibroblast (casein+5110 cells) of genetic modification; With

Embryo survival in the complete pregnant process of cloning transgenic cow embryo that Fig. 6 represents to rebuild with the old and feeble female fetal fibroblast (561 cell-line) of not breeding;

Detailed Description Of The Invention

The present invention relates to the improvement to nuclear transfer cloned animal embryo's prior art. Although consider Can be used for multiple mammal and other animal species to embryo cloning method of the present invention, but That this method is described with reference to bovine. Substantive characteristics of the present invention is that donorcells nuclear is in The G1 phase, preferably, it is early stage to be in G1.

A method from free growing cultured cell group, determining to obtain G1 phase cell be from Mitotic cell is selected on the culture surface one by one, and they are placed on comprise 10% tire ox blood Make it finish mitosis in the culture medium of (FCS) clearly. This donorcells is having silk to divide then In short period after splitting and entering S fusion receptors cell (being cytoplasm) before the phase, usually should With in this time be three hours, can detect with the 5-bromouracil deoxyribose mark. By this way, Cell is guaranteed to be in G1 in early days and is had the DNA of 2C content. So, the cell of circulation Before entering the G1/S separation, it is used for nuclear transfer. The cell of selecting is in the whole process of operation Leave in the culture medium of high serum content, after finishing with cytoplasmic fusion. The institute With, cell can not be induced and withdraw from CDC, can not be still in any meaning constantly.

Although the present invention considers to produce the synchronization G1 phase cell mass for nuclear transfer, this An advantage is needn't use to have cell genotoxic potential or interference cell synchronization in the bright method Reagent for example nocodazole or colchicine, for example, in advance with the cell of higher proportion with Stepization discharged from this retardance subsequently in the M phase again, waited that to select G1 after the cell division early stage Cell. But a large amount of G1 phase cells or invertibity ground will be for nuclear transfer in order to select G1 phase cell suppresses in specific time point, according to the present invention, with suitable drug concentration and incubating It is that cell cycle inhibitor is useful that the time of educating is used suitable reagent. This reagent and method Dawn is known to those skilled in the art. These methods can be included as and reduce the drug exposure time Pre-synchronization process, for example before secondary adds serum, use the serum starvation method inducing cell temporary The time enter the G0 phase, allow cell again enter G0/G1 separation or restriction point and carry out cell minute Split the cycle. Be fit to invertibity cell be suppressed at G1 phase different time points reagent (reference, Gadbois etc., 1992) comprising: (1) staurosporin (a kind of non-specific kinase inhibition Agent is in the extremely low concentration use of nanomole level); (2) act on the more special of cell cycle progression One inhibitors of kinases, for example cAMP dependent protein kinase and cGMP dependence protein swash Enzyme inhibitor; (3) Lovastatin; (4) isoleucine lacks; (5) dwell rhzomorph or hydroxyl of aphid The base urea uses in the mode that stops cell to enter the S phase, is arrested in the G1/S separation.

Because G1 phase donorcells nuclear contains the DNA (being that it is dliploid) of 2C content, it Can with have or the cytoplasm of high or low content MPF is rebuild. This that is to say, the G1 phase Nucleus can be before activate taking place, introduce afterwards or simultaneously cytoplasm. But, in order to restrain Grand embryo better grows, and preferably, donorcells nuclear (is merged at electric inducing cell no matter be Or directly introduce behind the nuclear injection) the cytoplasm of enucleation oocyte exist because of Expose reasonable time in the son, in order to promote nucleus to rearrange. This is referred to as " merging before activating " Perhaps FBA. Research work has in the past shown and basically has been " merge simultaneously and activate " Or AFS compare benefit (Stice etc., 1996 of this method; Wells etc., 1998; 1999). And, advise that open-assembly time is at least greater than one hour, preferably at 3-6 in cytoplasm Between hour, in order to improve the ratio of growing to blastocyst stage. But, when using this method, for In final embryo, keep correct times sex chromosome, stop micronucleus to form with appropriate method to be Important, wherein (Czolowska etc., 1984) take place in micronucleus when merging early than activation.

To rebuild and produce cloning with the G0 phase of cultivating or G1 phase donorcells in the bovine below Embryo's method outlines. In following detailed embodiment, collect the ox follicle cell from ovarian follicle (use of fibroblast is explained in embodiment 4-7 in embodiment 1-3 to use Bearing performance Bright). In actual applications, use present technique to prove and in essence anyly possess normal diploid The cell type of caryogram is all-round, comprises embryo, fetus, childhood and becomes body cell, no matter It is being bred or can induce and enter cell cycle or aging course. And, this area The additive method known in the art that the technical staff awares can be used for restructuring and produce gram Longhua embryo.

The maturation in vitro of egg mother cell

Collect the cow ovary in the slaughterhouse, put into physiological saline (30 ℃), transport in 2 hours The laboratory. Thereby the ovarian follicle that sucks the 3-10 millimeter with No. 18 entry needles and negative pressure reclaims ovarian cumulus one ovum Mother cell complex (COCs). (alternatively, the immature egg mother cell can be logical from the donor cow Cross ovum and extract collection, carry out subsequently maturation in vitro). COCs be placed to replenished 50 micrograms/ Milliliter heparin (Sigma, St.Louis, MO) and 0.4%w/v bovine serum albumin (BSA) The N-2-ethoxy piperazine of (Immuno-Chemical Products (ICP), Auckland, New Zealand) Piperazine-N '-2 ethane sulfonic aicd (HEPES) buffering tissue culture medium (TCM) 199 (H199; Life Technologies, Auckland, New Zealand) in. Before carrying out maturation in vitro, only select to have The COCs of tight non-locking ovarian cumulus corona radiata and homogeneous archiblast. These COCs train with H199 earlier Support base+10% hyclone (FCS) (Life Technologies, New Zealand) and wash twice, again Tissue culture medium (TCM) 199+10%FCS with buffered with bicarbonate washes once. Ten COCs are turned to Enter in this culture medium of 10 microlitres, be added to and place 5 centimetres of culture dishes (Falcon, Becton Dickinson Labware, Lincoln Park, New Jersey) 40 inner microlitre maturation medium In, cover with paraffin oil (Squibb, Princeton, New Jersey). Maturation medium comprises Replenish 10%FCS, 10 mcg/ml sheep follicular stimulating hormone (FSH) (Ovagen; ICP), 1 Mcg/ml sheep lutropin (LH) (ICP), 1 mcg/ml estradiol (Sigma), Tissue culture medium (TCM) 199 with 0.1 mM/l of cystamine (Sigma). The droplet culture dish 39 ℃, Moistening, contain 5%CO₂Gaseous environment in cultivated 18-20 hour. After the maturation, with COCs Place and contain 0.1% hyaluronidase (from bull testis; Sigma) HEPES buffering is synthetic Fallopian tubal liquid (HSOF; Thompson etc., 1990) eddy current was processed 3 minutes, used again HSOF+10%FCS washes three times, removes the ovarian cumulus corona radiata fully.

The nuclear transfer of cultured cell

A) egg mother cell of culture medium maturation, cytoplasm and reconstruction embryo after maturation until pulse In the cultivation based on H199 in the time section that fusion assessment in 15-30 minute finishes after the electric shock Preserve in the base or operate. Donorcells and M II cytoplasm were merged in 3-6 hour before activating The embryo that (FBA facture) rebuilds is synthetic at the AgResearch that deducts calcium+10%FCS Fallopian tubal liquid culture medium (AgR SOF; This culture medium is Gardner etc., and 1994 is described The modification prescription of prescription, by AgResearch, Hamilton, New Zealand has realized commerce Change) in cultivate, until activate the eve, namely cultivate 3-6 hour after. After this, calcium is present in all In the culture medium prescription that uses.

B) stoning with 15-20 micron (external diameter) glass pipette get except ripe about 18-20 of processing little The time the nucleus of oogonium, method is to draw first polar body and mid-term in the cytoplasm around Plate. Oogonium is containing 10%FCS earlier, and 5 mcg/ml Hoechst 33342 and 7.5 are little Grams per milliliter cytochalasin B (Sigma) dyeing 5-10 minute is not having Hoechst 33342 then This culture medium in operate. The stoning operation is confirmed by see nucleus under ultraviolet light. After the stoning, gained cytoplasm is extensive washing in H199+10%FCS, is kept at then this Plant in the culture medium, until inject donorcells.

C) preparation of static donorcells (being G0 phase cell) is cultivated with the method for serum disappearance Follicle cell be induced and enter resting stage (Campell etc., 1996b). The some day that routine goes down to posterity, Culture medium is sucked away, and with fresh PBS (PBS) cell is washed three times, adds again The fresh culture that only contains 0.5%FCS. Be put in the low blood serum medium follicle cell further Cultivate 9-23 days (common 10 days), be used for then nuclear transfer. Just at the injection eve, use standard Trypsin Induced prepares the donorcells single cell suspension. Sedimentation cell is resuspended in H199+0.5% Among the FCS, be kept in this culture medium until injection.

D) preparation of G1 phase donorcells (is for example added 10%FCS's with suitable culture medium DMEM/F12) the glass cover slide in culture dish is cultivated the follicle cell of propagation. From training Support in the ware and select the long cover glass that cell is arranged in its surface with sterile working, be placed on and carry out cell Or in the microoperation chamber of the suitable constructions of nucleus collection and injection. One droplet is comprised 10% The HEPES buffer culture medium of FCS drops on the cell, covers with mineral oil then. As long as cover glass Cell concentration is not too high on the sheet, just can distinguish and with the operation suction pipe carefully physically from Picking mitotic cell on the cover glass, because in the mitosis, cell rounding and only loose Loose ground is attached to culture surface. If this operation is not easy, comprising of dilution can used 1.5 (for example cultured cell system commonly uses transferred species to the insulin solutions of mcg/ml cytochalasin B Cultivate intensity 1/10th) front earlier with the very fast cleaning cell of PBS, mainly reduce from cover glass The mechanical damage that upper physics picking cell causes. Use suitable microscope (for example to differ micro-Mirror or DIC light microscope), mainly by seeing in the spindle that cohesion chromosome or identification are in Mitosis telophase, is still by the identification of the cohesion chromosome in the cell doublet of cytoplasmic bridge connection lid Mitotic cell on the slide. So, needn't use the DNA selectivity such as Hoechst Fluorescent dye also is exposed to cell in the ultraviolet light. Dependence is fixed on the injection dropper on the operator, Mitotic cell is individually chosen from cover glass, puts into the 10%FCS that comprises that closes on HEPES buffer culture medium droplet in, in order to finish mitosis and final cell division shape Become two cells. The diameter of suction pipe should be suitable, and it depends on clone, so that in operation Damaging cells or spindle that can physical property. So, preferably, be in later stage or latter stage Mitotic cell is selected, is taken out and allow to finish mitosis and be divided into two by single. These The paired cell that division produces can be exposed to by the short time the easy side of suitable enzymes solution then Method is separated into individual cells lightly. Then, each intact cell is injected into and is fused to carefully In the kytoplasm. Alternatively, nucleus can separated and directly be injected into the thin of enucleation oocyte In the kytoplasm. Preferably, little from three of beginning from culture surface picking mitotic cell at first The time in, finish donorcells nuclear transfered cell matter. This just guarantees that cultured cell is in the cell cycle G1 early stage and takes place in the S phase before fusion. For each cell type used herein or thin Born of the same parents system for example should use, and selects the negative 5-bromouracil deoxyribose labelling method of cell sample, confirms. And, the cell that suggestion confirm to use this method to select in fact be in the cell cycle and Sometime point in the back enters the S phase.

E) microinjection recipient cell kytoplasm is at the H199 culture medium that comprises 10%FCS and 5% sucrose Middle dehydration. This culture medium is also as the microoperation culture medium. Hold the suitable big of donorcells Little suction pipe (for example external diameter 30-35 micron) stung oolemma, cell by wedge at oolemma and cell Between the cell membrane of matter, with the cell membrane close contact that promotes to help to merge subsequently. After the injection, Rebuilding the embryo dewatered again through two steps: at first in the H199 of 10%FCS and 2.5% sucrose training Support in the base and dewatered 5 minutes, in H199+10%FCS, dewater until merge then.

F) Fusion of Cells was processed G0 phase and G1 phase cell, and the embryo uses the FBA strategy (merging before activating) rebuilds. After maturation begins about 24 hours (hpm), rebuild embryo Tire is by 0.3 mol/L sweet mellow wine, 0.5 mM/l of HEPES and contain 0.05 mM/ The buffer solution that the BSA of 0.05 FAF (FAF) of liter calcium and 0.1 mM/l of magnesium forms In carry out that electricity merges. Fusion is at room temperature, has two at one and covers with the fusion buffer solution Lid carries out in the cell of 500 microns stainless steel electrodes. To rebuild embryo and very thin with hand The alignment of mouth control pasteur pipet is so that the contact surface of cytoplasm and donorcells is parallel with electrode. For follicle cell, Fusion of Cells is by BTX electricity cell manipulation instrument 200 (BTX, San with twice Diego, the California) (needing to distinguish the appropriate electrical parameter of every kind of clone) emission, each 15 microseconds The pulse direct current of intensity 2.25-2.50 kv/cm is induced. After the electro photoluminescence, use H199+10% The embryo is rebuild in the FCS washing. Then in 15-30 minute, with micrography check fusion situation.

Do not expect that above-mentioned electrofusion parameter produces aobvious to the young cell matter of using at 24hpm The activation ratio of work, because after same electrical stimulates, age-grade contrast egg mother cell (n=112) only be lower than 1% formation protokaryon (Wells etc., 1999). This is to the FBA place Manage very importantly, so that NEBD and PCC take place, thereby allow G0 phase and G1 phase nucleus Be exposed to for Autosome and make chromatin reconstruct and nucleus heavy in the factor in the egg mother cell matter Compile.

Selectively be, as known to a person skilled in the art, from the cell of G1 phase cell Endorse with separated and be injected directly in the egg mother cell cytoplasm.

G) activate the method that multiple realization artificial activation is arranged. It is mould that a kind of method of uniqueness relates to ion Plain (Sigma) and the fast quinoline (6-DMAP of 6-dimethylamino; Sigma) (Susko-Parrish etc., 1994). After the fusion, the embryo preferably was exposed to egg mother cell matter 3-6 hour at donorcells nuclear After be activated. This preferred method is to be referred to as " merging before activating " (FBA; Wells etc., 1998). In activation front 30 minutes, (contain with HSOF with the fusion embryo of FBA processing method gained Calcium)+1 mg/ml FAF BSA washing is also preserved. Dripping 30 microlitres 5 micromoles per liter Among the HSOF+1 mg/ml FAF BSA of ionomycin (Sigma), 37 ℃, hatch 4 Minute induce activation. Activate and usually between cytoplasm age 27-30hpm, take place. Use HSOF+30 Mg/ml FAF BSA with embryo's washing 5 minutes, is containing 2 mM/ls extensively again The fast quinoline (6-DMAP of 6-dimethylamino; Sigma) AgR SOF (adding calcium)+10%FCS The middle cultivation 4 hours.

As FBA method (Stice etc., 1996:Wells etc., 1998; 1999) described in, Improve must be with suitable for the embryonic development ratio due to prolonging the time that nucleus is exposed to egg mother cell matter When prevention postpone to activate after micronucleus form the processing side of (Czolowska etc., 1984) generation The method combination. Serine-threonine kinase inhibitor, for example 6-DMAP, seemingly suitable examination Agent. So after initial activation stimulated, 6-DMAP allowed to form single intact cell nuclear, Thereby in the reconstruction embryo, kept correct ploidy.

The in vitro culture nuclear transfer embryo

Embryo Culture be the 20 microlitre AgR SOF that cover at mineral oil (can be from AgResearch, Hamilton, New Zealand buys) in carry out. AgR SOF is that SOFaaBSA (comprises 8 millis The FAF BSA of grams per milliliter; As Gardner etc., 1994 describe) the modification prescription. As long as May, nearly 10 embryos cultivate in the culture medium droplet together. The embryo is the module in moisturizing Hatch in the cell (ICN Biomedicals, Aurora, Ohio) 39 ℃ of cultivations in the admixture of gas that is formed by 5%CO2,7%O2 and 88%N2. 4-5 days that grow (0 day=The embryo rebuilds the same day), the embryo change over to 20 microlitres fresh be added with 10 micromoles per liter 2,4-dinitro Among the AgR SOF of phenol, WO 00/38538 is disclosed such as the publication specification, wherein 2,4-DNP has improved external ox embryonic development as the uncoupler of oxidative phosphorylation, will This specification is quoted from this as a reference. After the fusion the 7th day grown special to the portable blastocyst Levy and estimate.

Embryo transfer, pregnancy diagnosis and calving

Use technology well known in the art to carry out embryo transfer, pregnancy diagnosis and calving management.

Continuous kernel is transplanted and is cloned

In other embodiments of the present invention, may expect to use G1 phase donor thin to originating from Born of the same parents and suitable recipient cell are rebuild the cloning embryo's who produces the first generation and are carried out again time cloning. Time cloning can be by being separated into individual cells with PIE again, then with them each with Suitable recipient cell kytoplasm merges to be realized. As those skilled in the art perceive, The embryonic cell of this form (or egg division ball) clone requires donorcells and recipient cell The coordination of cell cycle in order to avoid chromosome undesired, makes the growth optimization. Preferably, Donorcells is to obtain at first generation cloning embryo's morula stage (about 32 cells of bovine) , but the early stage or late period that the embryo also can grow. Alternatively, can use from training earlier The G1 phase donorcells of supporting produces a clone fetus, and then obtains fetal cell system, uses This New cell line is clone embryos again. This again cloning process can be by prolonging former early stage in implantation The time that beginning donorcells nuclear is exposed to egg mother cell matter rearranges to nucleus and facilitates. This The bright original embryo's propagation clone embryos quantity generation second generation, the 3rd that also can use from the first generation In generation, waited among the cloning embryo and has advantage.

Continuous embryo transfer relates to nucleus and transplants continuously to the recipient cell kytoplasm environment that is fit to. Just Described in embodiment of the present invention, expectation earlier with G1 phase donorcells with to contain MPF higher Un-activation recipient cell kytoplasm rebuild a unicellular embryo. This will allow to take place NEBD and PCC phenomenon and activate when stimulating when catching up with subsequently, complete diploid cell nuclear will form. This nucleus (this nucleus has experienced nucleus reconstruct and nucleus is to a certain degree rearranged) Can from the single cell clone embryo, be used as the nucleus sucking-off then, be transplanted to subsequently to be in and be subjected to In the stoning embryonated egg in smart rear suitably period. Allow then embryonic development go on. This order The continuous kernel migration process can improve nuclear rearrange. This also may produce to grow and improve, because the Secondary nuclear transfer step is nucleus to be incorporated into the sperm fertilization impel it to be more suitable for being subjected to activating Enter in the cytoplasm environment of embryonic development.

The generation of transgenic animals

Doing nuclear transfer with the donorcells nuclear that is in the cell cycle G1 phase through genetic modification produces Breeding transgenic livestock may be a kind of ratio to embryonated egg procaryotic injection DNA (Wall etc., 1997) or Relate to injection sperm and the DNA Sperm-mediated transgenosis that fetters foreign gene in the cytoplasm (Perry etc., 1999) more effective general method. The nuclear transfer of making of cultured cell excellent Point comprises: the scope of (1) genetic manipulation may be more extensive; (2) with respect to collecting oogonium Or embryonated egg, make with clone that genetic manipulation is easier to be carried out in high genetic background; (3) institute The clone offspring that generation is arranged is genetically modified and has the sex of expectation; (4) and procaryotic injection Method or Sperm-mediated transgenosis produce single raw animal and compare, and this method has an opportunity lacking Produce direct drove and the herds that produce useful products in phase.

Cultured cell can be by inserting, removing or change suitable DNA and carry out hereditary change. Bring out radom insertion (this sequence can be allos), DNA that variation comprises new dna sequence dna Locus specificity inserts and allows the dna sequence dna of specific site in the genome to insert, lack or change The homologous recombination that becomes. Use methods known in the art to carry out cell selection and DNA analysis After having confirmed suitable hereditary change, can select then carefully from the normal transgenic cell of caryogram Born of the same parents are in the G1 phase cycle, are used for nuclear transplantation in order to produce cloning/transgenic animals.

According to specific gene difference in the operation, it is biomedical to exist multiple suitable genetic modification to change With the factor of agricultural with domestic animal. The scope of factor comprises; (1) in milk, blood or urine, produces medicine Use albumen; (2) produce nourishing product and medical food, for example milk; (3) control agricultural The characteristic of producing for example improves the quality and quantity of milk, meat and fiber and improves disease and evil The resistance of worm; (4) manufacture albumen is for example in milk; (5) heterograft; (6) Generation is as the domestic animal of human diseases model, for example cystic fibrosis and Huntington disease.

Therapeutic cloning

A remarkable influence of nuclear transplantation and embryonic stem cell technology may be based on the treatment field (Pederson, 1999) of people's cell.Specified disease takes place or can not repair or the tissue damage that can not self effectively upgrade (for example diabetes, amyotrophy, spinal cord injury, particular cancers, multiple nervous system disorders, comprise Parkinson's) patient have the potential that produces self treated tissue be used to transplant, long-acting or lifelong treatment is provided.At first this method is with end user's nuclear transplantation.This relates to from ill or injured patient collects a health tissues sample on one's body, and cultivation stimulates cellular proliferation.G1 phase donorcells by selecting the cell cycle and with they and the suitable recipient cell fusion cell nucleus of may rearranging.If recipient cell is the human oocyte of stoning, so, after suitable activation stimulated, the embryo of reconstruction can grow into blastocyst stage in suitable embryo culture medium.Under suitable condition of culture (Thomson etc., 1998), the human embryo stem cell can be from the internal cell populations of this clone embryos, and this cell will be the same in heredity with the patient of this cultured cell of donations.

From " embryonic stem cell " literal meaning, be meant any multipotency cell type of separation in the embryo, preferably separate internal cell populations from blastocyst stage with the present technique field method known to widely.

To be (the having the potential that is divided into any cell type in the cell) of undifferentiated, multipotency and possess external unlimited multiplication capacity in essence with the embryonic stem cell that the inventive method produced.Separation also has differentiation to a certain degree and possesses the limited differentiation capability that is divided into the various kinds of cell type from the cell-line of internal cell populations, but may still have therapeutic value.Experience according to the mouse embryonic stem cell, the condition that is fit to can develop, this condition can produce the purifying group of specific differentiated cell types, for example nerve cell, haemocyte, cardiac muscle cell etc. treat specific disease (for example producing the cell therapy diabetes of insulin or the cell therapy Parkinson's of generation dopamine).These heredity are gone up compatible cells and can be used back patient then, so as after transplanting the in-situ regeneration normal structure.Because cell is identical with patient in heredity, thus will can not be excluded, thereby needn't or need immunosuppressive drug hardly.Through system modifier gene seat, the major histocompatibility complex gene of in immune external cell recognition, playing an important role for example, it may be useful producing " general " embryonic stem cell line that is used for allograft.

Some application may relate to differentiation and transplant preceding genetic modification embryonic stem cell.This may be because gene therapy purpose is to transmit the medicine for treatment thing or correct for example dystrophin gene in the Duchenne muscular dystrophy patient bone flesh of somatic gene defect.

Except carrying out the transplantation treatment with cell, tissue or organ, human embryo stem cell is divided into particular cell types, and also the toxicologic study to drug discovery and human medicine is useful.

And, can be from cloning non-human animal offspring isolated cell, tissue and organ, be used for the transplanting of the human patient of this treatment of needs (external source transplanting).When this cell, tissue and organ comprise transgenosis, this cell, tissue and organ can be used for gene therapy or regulate the immune response of the external source tissue of patient.

Recipient cell during human body is used

In the method according to nuclear transplantation of the present invention, preferred acceptor is to use the enucleation oocyte of disclosed method preparation.Yet for human body was used, this was difficult.

A kind of substituting source of the nuclear recipient cell of clade of can rearranging is an embryonic stem cell.So, need a certain form cell therapy patient's somatic cell in cultivation, to dedifferente, and need not to be the human body egg mother cell.This can be by will being in the cell cycle G1 phase healthy somatic cell and stoning embryonic stem cell or one group of stoning embryonic stem cell (for a large amount of cytoplasm that are provided for rearranging) merge and realize.The cell cycle state of control acceptor stem cell is necessary, preferably is in M phase or G1 phase when merging.In this method, somatic noble cells is endorsed to dedifferente after being exposed to stem cell matter.Rebuild the cell that produces and to have the developmental potentiality versatility, may induce the cell type of one group of other specialization that produces treatment usefulness.In the heterozygote cell of thymic lymphocytes and embryonic genital cell fusion generation, confirmed (Tada etc., 1997) before this idea.

Specify the present invention with the following examples now, these embodiment as known to a person skilled in the art be not in order to limit the scope of the invention.

Embodiment 1The synchronization of folliculus donorcells is the ectogenetic influence after to nucleus transplantation in G0 phase or G1 phase

In this particular experiment, the primary cell line of folliculus granular cell is used for nucleus transplantation.This cell-line represents that with J1 it is from a New Zealand Jersey heifer.The J1 cell that is used in this experiment is that 7-8 in cultivating is for cell.

Donorcells is synchronized in two stages of cell cycle so that relatively:

(1) G0 phase cell is to cultivate in comprising the medium of 0.5%FCS, obtains in 10-11 days in the serum shortage.

(2) G1 phase cell is to merge with cytoplasm in after finishing mitosis 1-3 hour.

Negative 5-bromouracil deoxyribose mark detects and confirm that the J1 cell that merged does not enter the cell cycle S phase in mitotic 3 hour, but the G1 phase of cell cycle.

Using in this experiment and rebuilding the embryo from the donorcells of these two kinds of cell cycle processed group is culture in vitro in the AgR SOF medium that has replenished bovine serum albumin (BSA, Life Technologies production code member 30036-578).

Ectogenetic the results are shown in Table in 1.When electricity consumption was merged, cell cycle G0 phase (contrast) and G1 phase donorcells equated with the fusion efficiencies of cytoplasm (recipient cell).Shown in following table 1, the ratio that G0 phase and the ectogenesis of G1 phase donorcells processed group are rebuild the embryo to the fusion of blastocyst stage does not have difference.

Table 1: the cloning embryo's who rebuilds, in the AgR SOF medium that has replenished Life Technologies bovine serum albumin (BSA), cultivates with the J1 follicule cell that is in cell cycle G0 or G1 phase ectogenesis.Stage fusion rate 1-2 level total developmental rate G0 of blastocyst stage (n=152) 86 of cell cycle ± 5.2% 24 ± 1.1% 77 ± 6.4%G1 (n=186) 78 ± 5.0% 25 ± 6.2% 77 ± 6.9% Embodiment 2Folliculus donorcells (J1 and EFC cell-line) synchronization is in cultivating in the medium that has replenished the Sigma bovine serum albumin ectogenetic influence after G0 phase or the nucleus transplantation of G1 phase pair cell

Further evidence provides in table 2, so that support the folliculus donorcells that is in cell cycle G0 phase and G1 phase that the cultivation of culture in vitro clone embryos is grown after 7 days to not influence of blastocyst stage.

Experiment with two kinds independently follicule cell system carry out:

(1) " EFC "; A kind of from New Zealand's Fresian follicule cell system and

(2) " J1 " is a kind of plants small dairy from west, New Zealand pool.

In this experiment, the EFC cell is only used the G0 phase, and the J1 cell is only used the G1 phase of cell cycle.But the data of present embodiment gives comprehensively, because two kinds of cell-lines are cultivated on identical culture medium prescription from follicule cell and reconstruction embryo.In these experiments, medium is the AgR SOF medium of standard, but has replenished bovine serum albumin (the SigmaChemical Company of Sigma; Production code member A-7073) rather than as above-mentioned embodiment 1 in the bovine serum albumin of LifeTechnologies.

The J1 cell is used for nuclear transplantation during for passage cell at 3-6, and donorcells is to merge in after mitosis is finished 1-3 hour.The EFC cell is to use between generation and use between cultivating 9-18 days at the 3-8 that cultivates to contain 0.5% serum synchronization in the G0 phase.

Ectogenesis the results are shown in Table 2, shows for the folliculus donorcells, and growing between the phase to the fusion embryo's of blastocyst stage ratio at G0 phase of cell cycle and G1 does not have difference.

Table 2Stage fusion rate 1-2 level total developmental rate J1 of blastocyst stage G1 (n=576) 82 ± 3.8% 38 ± 3.6% 67 ± 1.8%EFC G0 (n=1108) 78 ± 2.3% 37 ± 2.3% 59 ± 3.3% in the ectogenesis cell line cell cycle of the clone embryos of rebuilding and in having replenished the AgR SOF medium of Sigma bovine serum albumin, having cultivated with the follicule cell that is in cell cycle G0 phase or G1 phase

Embodiment 3After the nucleus transplantation, the influence that the synchronization of folliculus donorcells was grown in the body after to nucleus transplantation in G0 or G1 phase.

The whole gravidic survival rate of clened cows embryo after being transplanted to receptor cow that data representation among Fig. 1 is rebuild with G0 that is in the cell cycle or G1 phase follicule cell.Data among Fig. 1 are to gather from embryo's data that top embodiment 1 and 2 illustrated experiments produce.This comprises the clone embryos that produces from two kinds of follicule cell systems, i.e. J1 and EFC.And, comprise in the same AgR SOF culture medium prescription of bovine serum albumin of the bovine serum albumin that replenished Sigma or Life Technologies producing clone embryos.These data gather to come in, because the bovine serum albumin in two kinds of follicule cells system or two kinds of sources is to not influence of the survival ability after transplanting (though having appreciable impact for growing to blastocyst stage).

Data representation among Fig. 1 is transplanted to 85 embryos that rebuild with the G0 donorcells of genital tract of synchronization receptor cow and 95 embryos that rebuild with the early stage donorcells of G1.

Fig. 1 illustrate with conventional ultrasound scanning, rectal palpation and calving the percentage of the fixed embryo/fetus that existed in the whole pregnant process of childbirth from the 7th day of embryo transplantation.Be to use the G1 follicule cell to cause the birth of the cattle on the hoof calf of giving a birth fully the most significantly.Compare with the G0 donorcells, from beginning in the 30th day of gestation until fully childbirth finish, have a declining tendency with the cloning embryo's of G1 cell reconstruction embryo survival, still, Bao Dao transplanting quantity does not reach statistical significance here.

For the G1 follicule cell, 9% transplanting embryo (9/95) causes the birth of the calf of giving a birth fully.But, in these calves 4 at birth or after the birth in the dust, the clone calf gross efficiency of living that causes producing from the G1 cell is 5%.On the contrary, G0 donor follicule cell causes 20% to grow childbirth (17/85) fully, because 3 calves are dead at birth, final gross efficiency of producing the calf that lives is 16% (14/85).

Embodiment 4With female adult SF (Age ± and Age-cell-line) synchronization after G0 or the nucleus transplantation of G1 pair cell, be supplemented with the influence of growing in the external and body of cultivating on the medium of Life Technologies bovine serum albumin.

Data come from two kinds of primary cell lines of adult SF independently.These two cell-lines are defined as " Age+ " and " Age-".They all are from the female cell system that selects the corresponding Angus beef cattle that adolesces sooner or later in ground.Since these two kinds of similar cell-lines in vivo with ectogenesis on significant not different, so data are compiled in collaboration with in following table 3 and Fig. 2.

Two stages of cell cycle compare in these experiments:

(1) G1, in the 1-3 after mitosis is finished hour, donorcells and cytoplasm merge.

(2) G0, donorcells was cultivated 3-12 days in the medium that replenishes 0.5%FCS.For the reconstruction embryo who is transplanted to receptor cow, cell is lacked by 4-5 days serum.

Cell from two kinds of cell-lines and two kinds of cell cycle processing is to substitute in nuclear transplantation in the 7th of cultivation.Rebuilding the embryo cultivates in the standard A gR SOF medium that has replenished Life Technologies bovine serum albumin (Life Technologies production code member 30036-578).

Listed data show the influence of the time span of G0 cell in low serum to fusion efficiencies in the table 3, still, in case just can ectogenesis subsequently not exerted an influence with the cytoplasm fusion, so G0 cytoplasm data are gathered.G0 and G1 cell cycle phase are to the not influence of growth to blastocyst stage.

Table 3With the female adult SF that is in G0 or G1 (Age+ and Age-cell-line) reconstruction, in the ectogenesis that is supplemented with the clened cows embryo who cultivates on the AgR SOF medium of Life Technologies bovine serum albumin.3-5 days (n=411) 66 ± 1.9% of total developmental rate G0 of stage fusion rate 1-2 level blastocyst stage of cell cycle ^a

19 ± 3.0% 58 ± 4.5% 12 days (n=107) 41 ± 6.9% ^bG1 (n=401) 59 ± 2.5%a ^b19 ± 5.0% 67 ± 6.6%

ab?P＜0.05

The data of Fig. 2 show that the G1 adult SF of selecting can produce the cattle on the hoof calf of childbirth fully.Similar to the data of Fig. 1, the trend of childbirth survival rate fully of existence is bigger than G0, still, does not have statistical significance here with regard to the transplanting quantity of carrying out.Use the G1 donorcells, the embryo of transplanting final 4% has produced cattle on the hoof calf (1/25).Compare, the G0 donorcells causes final 14% live embryo to grow childbirth (3/22).In this experiment, 4 of all productions calves have passed through postnatal development.

Embodiment 5Adult SF (LJ801 and 3XTC cell-line) synchronization after G0 or the nucleus transplantation of G1 phase pair cell, replenished in the body of cultivating in the medium of ICP bovine serum albumin and ectogenetic influence

In addition with two kinds independently the adult skin fibroblast line done the relatively experiment of G0 and G1 cell cycle phase.These two kinds of cell-lines are expressed as " LJ801 " and " 3XTC ".LJ801 is the male cell system from a Limousine X Jersey bull, and 3XTC is the hybridization cow from a kind of three generation trisome calves.

Two cell cycle phases give comparison:

(1) G0 cell, the donorcells of two kinds of cell-lines are to cultivate 4-5 days in containing the medium of 0.5%FCS; With

(2) G1 cell, in the 1-3 after mitosis is finished hour, donorcells and cytoplasm merge.

Negative 5-bromouracil deoxyribose tracer method confirms that the adult SF that merges in mitotic 3 hours does not enter the S phase.

LJ801 and 3XTC cell-line all are to substitute in nuclear transplantation at the 3-4 that cultivates to test.

Owing to do not have difference on the efficient of blastocyst stage and replenishing the identical standard AgR SOF medium of bovine serum albumin growing between LJ801 and the 3XTC cell from the reconstruction embryo of two kinds of cell-lines, but this time bovine serum albumin is from ICP (Immuno-ChemicalProducts, Auckland, New Zealand; Production code member ABFF-002).After the embryo transplantation, whole gravidic embryo survival is to be illustrated in Fig. 3 and 4 with corresponding cell-line 3XTC and LJ801.

The data of table 4 show that adult skin becomes the cell cycle synchronization of fiber donorcells not have to influence to growing to blastocyst stage at G0 or G1.

Table 4The ectogenesis of the clone embryos of rebuilding and in having replenished the AgR SOF medium of ICP bovine serum albumin, having cultivated with the adult SF that is in cell cycle G0 or G1 (LJ801 and 3XTC).Stage fusion rate 1-2 level total developmental rate G0 of blastocyst stage (n=145) 72 of cell cycle ± 7.8% 36 ± 6.1% 61 ± 5.0%G1 (n=200) 60 ± 3.1% 37 ± 6.0% 57 ± 5.0%

Data among Fig. 3 show that the embryo that rebuilds with 3XTC does not have difference to the 150th the sky surviving at least.For the 150th day embryo survival of the donorcells that is in cell cycle G1 is 25% (3/12), and the donorcells of G0 is 27% (3/11).

Data among Fig. 4 show rebuilds the embryo with the LJ801 fibroblast to exist the embryo that uses the G0 donorcells to survive trend the 210th day of gestation bigger, but this does not possess statistical significance.For the 210th day embryo survival of the donorcells that is in cell cycle G1 is 23% (3/13), and the donorcells of G0 is 39% (7/18).

Embodiment 6The influence that the bovine fetal fibroblast synchronization of genetic modification is grown in to external and body at G0 or G1 after the nucleus transplantation.

Also carried out the influence of the growth after the stage pair cell nucleus transplantation of research cell cycle with the female ox fetal lung fibroblast of genetic modification.Hereditary change comprises the insertion at random of the additional copies of ox β and к casein gene.Transgenic cell line is expressed as " casein+5110 ".

Two cell cycle phases give comparison:

(3) G0 cell, the donorcells of two kinds of cell-lines are to cultivate 3-6 days in containing the medium of 0.5%FCS; With

(4) G1 cell, in the 1-3 after mitosis is finished hour, donorcells and cytoplasm merge.

Rebuild the embryo and replenishing ICP bovine serum albumin (Immuno-ChemicalProducts, Auckland, New Zealand; Production code member ABFF-002) cultivates in the standard A gR SOF medium.

The ectogenetic data of embryo see Table 5.When the casein that is used for nuclear transplantation+5110 donorcellses are G1 during the phase, compare the G0 phase, grow to blastocyst stage and significantly reduced.Other cell-lines have formed contrast among this result and the former embodiment recited above.

Table 5Ectogenesis with the cloning ox embryo who is in cell cycle G0 or the female fetal lung fibroblast of the transgenosis in G1 stage (casein+5110 cell-lines) reconstruction and in the AgRSOF medium that has replenished the ICP bovine serum albumin, cultivates.Stage fusion rate 1-2 level total developmental rate G0 of blastocyst stage (n=150) 90 ± 8.1% 52 ± 1.8% of cell cycle ^a72 ± 2.9% ° of G1 (n=73) 77 ± 6.5% 26 ± 5.1% ^b43 ± 2.2% °

ab?P＜0.0b；cd?P＜0.01

Embryo survival data shown in Figure 5 show that for casein+5110 cell-lines the stage of cell cycle is to growing by the 90th day not influence at least.For the 90th day embryo survival of the donorcells that is in cell cycle G1 is 38% (9/24), and the donorcells of G0 is 27% (6/22).

Embodiment 7The influence of donorcells that do not breed, old and feeble (in the G1 later stage) to growing in the external and body after the nucleus transplantation

Done of the nucleus transplantation experiment of research ox donorcells that do not breed, old and feeble to development impact.The cell of using in this experiment is female fetal lung fibroblast and has passed through genetic modification (being labeled as 561 cells).Originally, cell is at active growth, and still, in incubation, it is slack-off day by day to grow, and to the later stage, cell enters the non-propagation phase, i.e. declining period known to the those skilled in the art of the present technique, cell division this moment stops.Senile cell is the G1 that is suppressed at the cell cycle, specifically at the separation (Sherwood etc., 1988) of G1/S phase.When cell enters declining period, in response to physiological mitogen, they are arrested in G1 late period, can not enter the S phase.So, be different from resting stage declining period, under latter event, cell can be induced once more and be entered the cell cycle, just can breed (for example adding serum in the cell culture that serum lacks) in case get back to appropraite condition.

The embryo that the transgenosis senile cell is rebuild cultivates in the AgRSOF medium that has replenished the ICP bovine serum albumin.

The data of extracorporeal embryo development see Table 6.Use old and feeble donorcells grow to the ratio of blastocyst stage be lower than that front embodiment illustrates to G0 or the early stage expected value of G1.

The clened cows embryo's that table 5 usefulness transgenosis fibroblast (561 cell) that do not breed, old and feeble rebuilds, cultivated in having replenished the AgR SOF medium of ICP bovine serum albumin ectogenesis.(n=158) of total developmental rate 561 agings of stage fusion rate 1-2 level blastocyst stage in cell line cell cycle 88% 12% 35%

The cloning efficiency of not breeding senile cell that the Notes of Key Data of embryo survival shown in Figure 6 is suppressed at G1 late period is low, has only 4% embryonic development to the 90 days (1/26).

Conclusion

1. select the individual cells of the certain phase of cell cycle, preferred G1 is early stage, is possible.This technology with respect to the so-called proliferative cell of former use has advantage, and in the former technology, stage and the inaccuracy of actual cell cycle that is used in the individual cells of nucleus transplantation known.

2. owing to produced the calf that lives behind the nuclear transplantation, be all-round so be in the early stage cell of the G1 of cell cycle after the mitosis.

So, and G0 be not with nucleus transplantation after unique stage of compatible cell cycle of growth, with the cultured cell and the G1 cell of differentiation, the early stage cell of G1 preferably, cell nucleus also can functionally be rearranged.

4. the early stage cell of the G1 after the mitosis promotes to grow similar level to blastocyst stage as the G0 cell after nucleus transplantation.

5. the cloning embryo with G0 or the early stage donorcells nuclear reconstitution of G1 does not have marked difference on the survival rate after the transplanting.

Commercial Application

The present invention may set up on cloning drove/herds of animal useful, comprise the albumen that can produce pharmaceutically useful, agriculturally useful product for example the transgenic animal and the human body of meat, milk and fiber use, particularly in clone's property treatment field.

Should be appreciated that this describes original idea is not to limit the scope of the present invention in the foregoing description, the many variations that it will be apparent to those skilled in the art that are possible, and do not depart from the scope of appended claims.

List of references

Barnes F L; Collas P; Powell R; King W A; Westhusin M and Shepherd D (1993). in nucleus transplantation ox embryo, the influence that the stage of acceptor egg mother cell cell cycle of living in is synthetic to DNA, nuclear is touched disintegration, chromosome reorganization and grown. molecule reproduction and auxology (Molecular Reproduction and Development) 36:33-41.

Boquest A C; Day B N and Prather R S (1999). fibroblastic cell cycle of pig tire that the flow cytometry analysis is cultivated. biology of reproduction (Biology ofreproduction) 60; 1013-1019.

Campell K H S; Loi P; Cappai P and Wilmut I (1994). with inferring that the stoning be in the S phase activates sheep nuclear transfer embryo that egg mother cell rebuilds and grows improvement to blastocyst stage. biology of reproduction (Biology ofreproduction) 50; 1385-1393.

Campell K; Mc Whir J; Ritchie B and Wilmut I (1995). produce the lamb that lives behind the embryonic disc nuclear transplantation of cultivation. animal genesiology (Theriogenology) 43:181.

Campell K H S; Loi P; Otaegui P J and Wilmut I (1996a). the coordination of cell cycle in the embryo cloning of use nucleus transplantation. reproduction comment (Reviews ofReproduction) 1:40-46.

Campell K; Mc Whire J; Ritchie W A and Wilmut I (1996b). carry out nucleus transplantation clone's sheep from cultured cell system. natural 380:64-66.

Cibelli J B; Stice S L; Golueke P J; Kane J J; Jerry J; Blackwell C; Ponce De Leon F A and Robl J M (1998). produce the transgenosis calf of cloning from the nonstatic fetal fibroblast. science 280:1256-1258.

Collas P; Balise J J and Robl J M (1992a). the cell cycle phase of donorcells nuclear is to nucleus transplantation rabbit embryo's development impact. biology of reproduction (Biology ofreproduction) 46; 492-500.

Collas P; Pinto-Correia C; Ponce De Leon F A and Robl J M (1992b). the donorcells phase of the cycles is to nucleus transplantation rabbit embryo's chromatin and the morphologic influence of spindle. biology of reproduction (Biology ofreproduction) 46; 501-511.

Czolowska R; Modlinski J A and Tarkowski A K (1984). the behavior of thymic lymphocytes nuclear in the oocyte of mouse of un-activation or activation. cell science magazine (Journalof Cell Science) 69:19-34.

Gadbois D M, Crissman H A, Tobey R A and Brodbury E M (1992). G1 phase a plurality of kinase inhibition site of non-transformed mammalian cell is default in transformant. institute of American Academy of Sciences newspaper (Proceedings of the National Academy of Sciences, USA) 89:8626-8630.

Gardner D K; Lane M; Spitzer A and Batt P (1994). lack serum and somatic cell and improved the spilting of an egg of sheep fertilized egg and the culture in vitro growth ratio to blastocyst stage: the embryo in amino acid, vitamin and the cultivation has stimulated growth. biology of reproduction (Biology ofreproduction) 50; 390-400.

Pedersen R A (1999). medical embryonic stem cell. Scientific Beauty compatriots (ScientificAmerican) April, 1999:44-49.

Perry ACF Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y and Yanagimachi R (1999). carry out mammalian genes with sperm injection method in the cytoplasm and shift. science 284:1180-1183.

Schnieke A E; Kind A J; Ritchie W A; Mycock K; Scott A R; RitchieM; Wilmut I; Colman A and Campell KIIS (1997). produce the Transgenic Sheep that contains the people IX factor from the fetal fibroblast transplanted cells nuclear of transfection. science 278; 2130-2133.

Sherwood S W, Rush D, Ellsworth J L and Schimke R T (1988). in the IMR-90 cell, determine cell ageing: a kind of flow cytometry method. institute of American Academy of Sciences newspaper (Proceedings of the National Academy of Sciences, USA) 85:9086-9090.

Stice S L; Strelchenko N S; Keefer C L and Matthews L (1996). pluripotency ox embryo cell line instructs the embryonic development after the nucleus transplantation. biology of reproduction (Biology ofreproduction) 54; 100-110.

Susko-Parrish J L; Leibfried-Rutledge M L; Northey D L; SchutzkusV and First N L (1994). the moment calcium phenomenon of inducing causes the inhibition of protein kinase does not finish the transformation of maiotic ox oogonium to embryo's cycle. Developmental Biology (Developmental Biology) 166:729-739.

Tada M, Tada T, Lefebvre L, Barton S C and Surani M A (1997). in the heterozygote cell, the nuclear back of embryonic genital cell inductor is given birth to and is rearranged. European molecular biology magazine (The EMBO Journal) 16:6510-6520.

Thompson J G E; Simpson A C; Pugh P A; Donnelly P E and Tervit HR (1990). oxygen concentration is to sheep before implanting and ox embryo's ectogenetic influence. reproduction and fertilization magazine (Journal of Reproduction and fertility) 89:573-578.

Thomson J A, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, SwiergielJJ, Marshall VS and Jones JM (1998). the embryonic stem cell line that from people's blastocyst, obtains. science 282:1145-1147.

Vignon X; Chesne P; Le Bourhis D; Flechon J E; Heyman Y and Renard J P (1998). merge the ox embryo's who rebuilds potentiality of development .C R Academy of Science, Paris 321:735-745. with the egg mother cell of stoning maturation and the somatic cell of cultivation

Vignon X; Le Bourhis D; Chesne P; Marchal J; Heyman Y and RenardJ P (1999). with the nuclear transplantation in cattle embryo's of the SF reorganization of static and propagation growth. animal genesiology (Theriogenology) 51:216.

Wakayama T and Yanagimachi R (1999). born of the same parents clone the adult male mice from adult tail taper. natural genetics (Nature Genetics) 22:127-128.

Wall RJ, Kerr DE and Bondioli KR (1997). transgenosis milk cow: extensive genetic engineering. cow science magazine (Journal of Dairy Sceince) 80:2213-2224.

Wells D N; Misica P M; McMillan W H and Tervit H R (1998). use cell from fetal fibroblast system to carry out producing after the nucleus transplantation ox fetus of cloning. animal genesiology (Theriogenology) 49:330.

Wells D N; Misica P M; Tervit H R and Vivanco W H (1999). the adult body-cell neucleus transplanting is used to preserve last Enderby land kind cow. reproduction, fertilization and growth (Reproduction, Fertility and Development) 10:369-378.

Wilmut I, Schnieke A E; McWhir J; Kind A J and Campbell K H S (1997). from mammal fetus and the offspring who becomes the somatic cell acquisition to live. natural 385:810-813.

Zakhartchenko V; Durchova-Hills G; Stojkovic M; Schenthaner W; Prelle K; SteinbomR; Muller M; Brem G and Wolf E (1999). serum starvation and again time cloning to the influence of the nucleus transplantation efficient of bovine fetal fibroblast. reproduction and fertilization magazine (Journal of Reproduction and Fertility) 115:325-331.

All references are comprehensively to quote from this to carry out reference.

Claims (55)

1. the method for a nuclear transplantation, this method comprise to be selected from donorcells group propagation or that do not breed and separates the G1 cell, and a nucleus transplantation that will obtain from the G1 cell of separation like this is in the stoning recipient cell.

2. the method for claim 1, wherein described donorcells group is the one or more known or unknown stage that is in the cell cycle.

3. method as claimed in claim 1 or 2, wherein, described donorcells group be do not breed and synchronization in any one time point in cell cycle G1 stage.

4. as each described method of claim 1-3, wherein, described G1 cell is in the early stage separation of G1.

5. as each described method of claim 1-3, wherein, described donorcells group be do not breed and comprise senile cell.

6. as each described method of claim 1-5, wherein, described donorcells group is from animal body or the embryo who separates the vitro cell culture, fetus, childhood or become somatic cell.

7. method as claimed in claim 6, wherein, described donorcells group comprises anyly can enter the normal cell of dliploid caryogram of cell cycle and propagation by irriate.

8. method as claimed in claim 7, wherein, described donorcells group is in the neoblast state or is in differentiation or arbitrary degree of resting stage or declining period.

9. each described method in the claim as described above, wherein, described donorcells is fibroblast or the follicule cell of adult or fetus.

10. each described method in the claim as described above, wherein, described donorcells comprises the modification cell.

11. method as claimed in claim 10, wherein, described donorcells comprises transgenic cell.

12. each described method in the claim as described above, wherein, described recipient cell comprises an enucleation oocyte.

13. method as claimed in claim 12, wherein, described enucleation oocyte is to obtain from the species consistent with donorcells nuclear source.

14. as each described method of claim 1-11, wherein, described recipient cell comprises a stoning stem cell or a stoning stem cell that merges.

15. method as claimed in claim 14, wherein, described stem cell is from the embryo who is growing or the embryonic stem cell that separates from a kind of cultured cell system that has set up.

16. a method of producing the cloned animal embryo, this method comprise the donor nuclei in the G1 stage that is in the cell cycle of a separation is transplanted in the recipient cell of stoning.

17. method as claimed in claim 16, wherein, described donorcells nuclear carries out genetic modification with the known method in present technique field so that produce the cloning embryo with desired genetic character.

18., when it is used to produce the cloning embryo of a kind of animal, be selected from: birds, amphibian animal, fish and mammal as claim 16 or 17 described methods.

19. method as claimed in claim 18, wherein, described cloning animal embryo is a kind of mammal, is selected from: comprise human primate, rodent, rabbit, cat, dog, horse, ox, sheep, deer, goat and pig.

20. reconstruction animal embryo with method preparation as claimed in claim 16.

21. the described reconstruction animal embryo of method as claimed in claim 17 comprises transgenic embryo.

22. one kind as claim 20 or 21 described reconstruction animal embryos, its again time cloning further increase embryo's quantity or it is transplanted continuously through cell nucleus, helper cell nuclear is rearranged and/or is grown.

23., comprise being selected from the mammal that comprises human primate, rodent, rabbit, cat, dog, horse, ox, sheep, deer, goat and pig as each described reconstruction animal embryo of claim 20-22.

24. a method of cloning the non-human animal, this method comprise the steps: that (1) allows the non-human animal embryo grow term from embryonic period, embryonic phase according to claim 16 or 17 each described method production cloning non-human animal embryos (2) use known methods; (3) arbitrarily breed the non-human animal who forms thus with conventional method or further PCR cloning PCR.

25. method as claimed in claim 24, wherein, described cloning non-human animal is selected from: the non-human mammal that comprises human primate, rodent, rabbit, cat, dog, horse, ox, sheep and deer.

26. as claim 24 or 25 described methods, wherein, it is a kind of transgenic nonhuman animal with expectation genetic character that described clone talks about the non-human animal.

27. method as claimed in claim 26, wherein, described transgenic nonhuman animal is a kind of transgenic cow or sheep.

28. cloning non-human animal with the preparation of method as described in claim 24.

29. cloning non-human animal as claimed in claim 28 comprises being selected from: the mammal of non-human primates, rodent, rabbit, cat, dog, horse, ox, sheep and deer.

30. as claim 28 or 29 described cloning non-human animals, comprising transgenic nonhuman animal with desired genetic character.

31. cloning non-human animal as claimed in claim 30 comprises transgenic cow or sheep.

32. as claim 30 or 31 described cloning transgenic animal, wherein, described desired genetic character is selected from: insert, lack or change one or more genes, it can produce pharmaceutical protein in milk, blood or urine; Produce nourishing milk meat product; Produce useful agricultural production proterties so that improve the quality that milk, meat and fiber are produced; Raising is to the resistance of insect and disease; Manufacture albumen in milk; Heterograft; And be used to produce transgenic animal as the human diseases model.

33. as claim 28-32 each described cloning non-human animal offspring and descendant thereof.

34. method that produces embryo cell line, this method comprises the steps: a) to select from donorcells propagation group or G1 cell synchronization cell mass or senile cell group separates the G1 cell, then from isolated cells like this in a nucleus transplantation to the stoning recipient cell; B) cultivate blastocyst stage; C) reclaim embryonic cell; And d) use method well-known in the art to set up external unlimited propagated cell system.

35. method as claimed in claim 34, wherein, described embryonic cell is an embryonic stem cell.

36. as claim 34 or 35 described methods, wherein, described donorcells is people's cell.

37. as each described method of claim 34-36, wherein, described donorcells and recipient cell all are people's cells.

38. as each described method of claim 34-37, wherein, described donorcells is adult or the fetal cell that is selected from the normal cell type of any caryogram, recipient cell is to be selected from any cell type of gene expression of can rearranging.

39. one kind with the embryo cell line of producing as each described method of claim 34-36.

40. a human embryonic stem cell of producing with method as claimed in claim 36 when according to claim 35, has using value on treatment is used.

41. method of producing embryonic stem cell, this method comprises the steps: a) to select from donorcells propagation group or G1 cell synchronization cell mass or senile cell group separates the G1 cell, then from isolated cells like this in a nucleus transplantation to the stoning recipient cell; B) cultivate blastocyst stage; And c) reclaims embryonic stem cell.

42. method as claimed in claim 41, wherein, described donorcells is people's cell.

43. as claim 41 or 42 described methods, wherein, described donorcells and recipient cell all are people's cells.

44. as each described method of claim 41-43, wherein, described donorcells is adult or the fetal cell of selecting from the normal cell type of any caryogram, recipient cell is to select from any cell type of gene expression of can rearranging.

45. use the embryonic stem cell that method is produced as described in each as claim 41-43.

46. embryonic stem cell as claimed in claim 45 is comprising human embryo stem cell.

47. purposes as claim 39,40 and 45 embryonic cell as described in each, wherein, specialized cell or the types of organization that is selected from nerve cell, muscle cell, heart cell, liver cell, pneumonocyte, kidney cell or other a kind of relevant cell types cultivates with method well-known in the art.

48. a purposes as claimed in claim 47, wherein, described embryonic cell is as claim 40 or 46 described human embryo stem cells.

49. therapeutic cloning method, wherein, embryonic stem cell is that each is described according to claim 35 and 41-43, produce from the recipient cell of curee's gained, and cultivate and produce the described curee that is used for this treatment of needs or other curees transplanting with specialized cell or tissue.

50. method as claimed in claim 49, wherein, described embryonic stem cell comprises one or more transgenosiss, gives the genetic character of the resulting noble cells expectation that is used to transplant.

51. a methods of treatment that is used for passing through disease, obstacle or the damage of transplanting specialized cell or tissue treatment, this method comprise specialized cell or tissue to the claim 49 of patient's administering therapeutic effective dose of this kind of needs treatment or 50 described methods productions.

52. as claim 49 or 50 described methods, wherein, described disease, obstacle or damage are selected from: multiple nervous system disorders (for example Parkinson's), diabetes, cardiopathy, amyotrophy, multiple hereditary disease, particular cancers (for example leukemia), spinal cord injury, burn and other miseries.

53. a vitro differentiation human embryo stem cell that uses the described method of claim 47 to produce carries out the method for drug screening or drug toxicology test.

54. a heteroplastic transplantation method, wherein, cell, tissue and organ are from as separating each described inhuman cloning animal of claim 28-32, and are used for the human diseases patient's of this treatment of needs transplanting.

55. a gene therapy method, wherein, cell, tissue and organ comprise transgenosis, and are separated and are used for as claim 30 or 31 described inhuman cloning animal embryos.

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