CN1377424A - Methods of reparing tandemly repeated DNA sequences and extending cell life-span using nuclear transfer - Google Patents

Abstract

This invention relates to methods for rejuvenating normal somatic cells and for making normal somatic cells of a different type having the same genotype as a normal somatic cell of interest. These cells have particular application in cell and tissue transplantation. Also encompassed are methods of re-cloning cloned animals, particularly methods where the offspring of cloned mammals are designed to be genetically altered in comparison to their cloned parent, e.g., that are 'hyper-young.' These animals should be healthier and possess desirable properties relative to their cloned parent. Also included are methods for activating endogenous telomerase, EPC-1 activity, and or the ALT pathway and/or extending the life-span of a normal somatic cell, and other genes associated with cell aging and proliferation capacity.

Description

Recover and the cell survival prolongation by the telomere in the animal of old and feeble somatic cell clone

The cross reference of related application

The application's book is the part continuation application of U.S. serial 09/527,026 and 09/520,879, and requires the right of provisional application 60/152,340 and 60/153,233.

Invention field

The present invention relates to stagnate (checkpointarrested), approaching aging or have the method for normal or the modification somatocyte or the cell DNA in undesirable short cell life-span by nuclear transfer technology rejuvenation aging, the outpost of the tax office.These methods are particularly useful for rejuvenation because the clone after the complex inheritance operation expands or chronic tissue injury has reached old and feeble or approaching old and feeble cell, thereby improve the ability that these cells are used to produce the cloned, transgenic animal as donor or are used for human cell's treatment.

The present invention also can be used for rejuvenation because ageing or because the illness relevant with the cell aging that increases the weight of (as muscular dystrophy or atherosclerosis, immunosenescence, BPH, neurodegenerative disease, the sclerosis of Barrett esophagus, AMD osteoarthritis and skin ulcer) and aging or aged cell gradually.Cell by nuclear transplantation or relevant technologies Reprogrammable or recovery patient or animal makes its regeneration and reverts to totipotency.These totipotent cells can be used for producing following cellular type, include but not limited to: multipotential cell, as a matter or preceding interstital stem cell, hematopoietic cell, vascular cell etc., they can be transplanted in patient or animal or the suitable donor.These cells will be in patient or animal tissues " sowing " but polytype health proliferative cell comprises bone, blood, muscle, neurone, immunocyte and other type.

Method of the present invention comprises that also this teratoma contains the cell from any or all three germinal layer, and can be used for preparing the dissimilar primary cell that the homologous genes type is arranged with the purpose primary cell by the cell of the cell of rejuvenation and teratoma generation differentiation.The primary cell of these new generations is significant in organizational project and organ transplantation treatment field.Also comprise clone clone's mammiferous method again, particularly compare that the descendant inheritting of cloning mammal is learned the method that changes with clone's parental generation.

The present invention also relates to identify the test of compound that adjustable ganglion cell is aging and old and feeble and associated gene, particularly can influence the compound of other index of telomere length, EPC-I activity, tPA, collagenase activities, gas gene, mitotic index and cell senescence and multiplication capacity.

Background of invention

Be characterised in that the marked improvement of clone's science over the past decade, witnessed clone sheep (i.e. " how sharp ") (Roslin Bio-Med), be named as the birth of " Mira " three clone goats (GenzymeTransgenics utilizes the technology of ACP permission), tens clened cows (ACT), number generation clone mouse and nearest 5 clone pigs (PPL).Clone technology also makes progress, make can be used for now from grow up, the nuclear cloning mammal of noble cells, scientists it is now know that their experience " reprogramming " in being imported into enucleation oocyte the time.Referring to United States Patent (USP) 5,945,577, be incorporated herein by reference.

Can be used for having great importance for organ, cell and tissue transplantation field from nuclear generation embryonic cell or this fact of embryonic stem cell of adult differentiated cells.For example, can produce embryonic stem cell, be induced to differentiate into the cellular type that transplanting needs by the nuclear of the cell of taking from the patient who needs transplanting.Utilize the technology of field of tissue engineering technology, can be by the design organization of clone's noble cells and the organ that can be used for transplanting.Because the cell that is used to transplant has identical nuclear gene type with tissue with the patient, will avoid or reduce the problem of transplant rejection and use the inherent danger of immunosuppressive drug institute.And engineering cell and organize available allogeneic dna sequence DNA easily to modify, or modify and make the deleterious gene inactivation makes the cell transplanted and be organized in the relevant or improvement of genetics in case of necessity.The United States Patent (USP) series number that has and apply for simultaneously with the present invention _ _ method of genetic modification donor nuclei DNA and acceptor Mitochondrial DNA has been discussed, this complete quoting as a reference.

Yet, some worries about the hereditary age of clone cell are arranged recently.People (Nature (1999) 399:316) such as nearest Shiels are about the many sharp reports promptings of clone sheep, perhaps, nuclear transplantation can not recover telomere length, and end limit fragment (TRF) size of finding in the animal by embryo, tire and adult cell clone has reflected the mortality ratio of the nuclear of transplanting.Relevant especially (people (1999a) NatureMed.5:975 such as Lanza of the implication of these discoveries with the clone who is used for human displacement cell of transplanting and tissue; People such as Lanza (1999b) Nature Biotechnol.17:1171).The transplant organ of experience prematurity aging will become destructive for surrounding tissue in the body, in fact can increase the weight of to replace the disease that cell will be treated.People's such as Shiels report has also proposed whether to experience about the cell that nuclear transplantation produces the cloned animal the problem whether life-span reduces of prematurity aging and nuclear transplantation generation.This to the clone of high quality domestic animal and again the clone have again and seriously influence, and thought that before this report the clone is better than traditional breeding technology, the latter depends on the animal that reached mating age before breeding another generation.

Over at least two ten years, scientists supposition telomere is lost relevant with weathering process.Referring to, Harley, " telomere is lost: the mitotic division clock still is hereditary time bomb? " Mutation Res. (1991) 256:271-282.This hypothesis is called as " marginotomy theory " at first, that is, losing gradually of end of chromosome or telomere causes the cell cycle to be withdrawed from, cell aging as a result.Referring to, Olovnikov, " Marginotomy theory " J.Theor.Biol. (1973) 41:181-190.This hypothesis has proposed archaeal dna polymerase the earliest owing to need the RNA primer to duplicate lagging strand, the therefore prediction of duplicated chromosome end fully.This prediction is confirmed by molecular studies at last, studies show that the segmental mean length of the chromosomal end limit of human fibroblasts reduces in external mode of duplicating with dependence.Referring to, people such as Harley, " telomere shortens in human fibroblasts's weathering process ", Nature (1990) 345:458-460.

Support that the further evidence of telomere theory is relevant with Telomerase.Telomerase activation in people's cell was identified for the first time in 1989.Referring to, Morin, " people's Telomere terminal transferase is synthetic a kind of ribonucleoprotein of TTAGGG multiple " Cell (1989) 59:521-529.Telomerase is used for setting up at end of chromosome, recovers telomere length.Other studies show that although telomerase activation is suppressed, Telomerase has activity in some stage that germ line cell duplicates in the somatocyte atomization, therefore keep telomere length in germ line cell between generations.In addition, the display end granzyme also has activity in transformant.Referring to, the summary of Harley (1991).

Once proposed, the inhibition of Telomerase may limit somatocyte is cloned expansion (as in cancer) in uncontrolled mode ability in the noble cells.But the immortality of some tumor cell line display end granzyme feminine gender, this is called as " ALT " approach.The inventor proposes, and this alternative route as the acquisition of tumour generation Telomerase Activity, is that kind is the reproduction of proterties.The inventor proposes, and impaired telomere is repaired in kind of system, is not only by Telomerase and adds the telomere repetition, and invade and extension by the homology chain that archaeal dna polymerase causes.

Because sexual propagation has been walked around in nuclear transplantation, promptly use the source of the somatocyte different as nuclear DNA with germ line cell, current hypothesis about the clone is, and clone's telomere is regenerated never, and cloned animal and its parental generation are same " the hereditary ages ".In fact, have been noted that in the certain hour before being used for nuclear transplantation relevant with this technology.Referring to, BBC news, " her age of many Libiees is older? " on May 27th, 1999, Thursday.If this theory is correct, will mean that clone's cell may have shorter mean lifetime than the animal of the same age that produces by sexual propagation, perhaps animal may have the life-span shorter than its parental generation.

This theory not only seriously influences for filed of organ transplantation, and the genetic manipulation degree that can carry out the somatocyte that is used for nuclear transplantation has been proposed suspection.For example, nuclear transfer technology major advantage is that the transgenosis transfection more easily be preserved and use to somatocyte can than embryonic stem cell in cultivation.This characteristic helps producing producing treats proteic animal, that is, for example, by the cow that allows in milk, to produce the proteic mammary gland specific promoter express transgenic of treatment.Equally, a series of genetic manipulations be can not stand owing to aged karyomit(e), then in fact can not animal, cell and tissue be produced with multiple genetic manipulation if be used for the cell of nuclear transplantation.Yet the ability of carrying out these complex inheritance operations may be necessary, for example, before these cells are used for nuclear transplantation and organ transplantation, corrects from the genetic abnormality in the donorcells of the patient with detrimental mutation.

Explain why some researchists find that telomere can't hypothesis of regenerated be after nuclear transplantation, telomere regeneration depends on the selection of donor somatocyte type.Nearest studies show that, the reconstruction of telomerase activation causes telomere to prolong and the infinite multiplication of normal people inoblast and retinal epithelium cell (people (1998) Science 279:349 such as Bodnar; Vaziri and Benchimol (1998) Curr.Biol.8:279), use the epithelial similar experiment of breast can not cause the prolongation of telomere and duplicate life-time dilatation people (1998) Nature 396:84 such as () Kiyono.Cell difference on the activated signal transduction path on the ability of Telomerase prolongation telomere or after adaptation is cultivated is used for explaining these differences (de Lange and DePinho (1999) Science 283:947).

Some researchists propose, and telomerase activation may be to rely on the cell cycle.For example, in 1996, Dionne reported that the telomerase activation cell is at (G stationary phase ₀Phase) downward modulation of telomerase activation, and the supposition telomerase activation may be to rely on the cell cycle.See http://telomeres, virtualave.net/regulation.html.Similarly, people such as Kruk have reported that when comparing with other time point of cell cycle the Telomerase (Biochem.Biophys.Res.Commun. (1997) 233:717-722) of higher level is arranged in early days at S.Yet other researchists have reported the result of contradiction with it, propose telomerase activation and growth velocity rather than relevant (people such as Holt, (1996) Mol.Cell.Biol.16 (6): 2932-2939 with the cell cycle in addition; Referring to above network address, with reference to Holt, 1997 and Belair, 1997).Other people propose in addition, and Telomerase activates by other cell-stimulating signal mediation, Telomerase response CD40 antibody/antigen receptors bind and contact the interleukin-4 rise and proved that (network address is above quoted Weng, 1997 to this point together in the external B cell; Referring to people such as Hiyama (1995) J.Immunol.155 (8): 3711-3715).Although but increasing to the interest of Telomerase and its effect in aging and cell conversion process, active adjusting is still known little to Telomerase.Referring to, for example, Smaglik, " turn to Telomerase: when the antisense strategy occurred, basic problem still existed " TheScientist, on January 18th, 1999,13 (2): 8.

Granzyme active ability in adjustable side has wide influence in medical circle, and may influence dearly than the telomere more technology of regenerating in the clone animal.Have the ability of regulating telomere can treat many ages relevant with disease other type.For example, the ability of adjusting telomere may be important for the usefulness that improves the bone marrow transplantation relevant with cancer chemotherapy; The Telomerase treatment can be used for replacing the aged cells in the immunity system, for example in eye retina, in the treatment vascular lamina, be beneficial to preventing heart disease outbreak or apoplexy, prolong the hepatocellular life-span, treatment liver cirrhosis, or prolong the myoblastic life-span, treatment muscular dystrophy.In addition, the ability of adjustable side granzyme can allow control cancer cell.At last, the model that the external model that telomere and Telomerase are regulated, particularly cell senescence reverse can design and identify that telomere is regulated, the test and the sieve method of the molecular mechanism of aging and cancer.Therefore, more understanding in depth of regulating of Telomerase except guaranteeing the usefulness of cloned tissue for organizational project and transplanting, and guaranteed even improves to clone and outside life-span of non-cloned animal, can also cause wide range of therapeutic.

Summary of the invention

The present invention is based on unexpected discovery, according to recently for the cloning mammal suspection at heredity age, the nuclear transplantation method can make aging or approaching old and feeble cell rejuvenation, and reparation (in telomere) series connection multiple dna sequence dna, recover the young pattern of genetic expression, as improve the EPC-1 activity, and/or improve cell survival or ability of cell proliferation.Therefore the invention enables according to thinking that for the worry of nuclear transplantation impossibility becomes possibility recently; Promptly, old and feeble or approaching old and feeble cell, for example at cell near incubation growth before old and feeble, or the cell that from human or animal, obtains with age related defects or illness, still can be used for producing clone's cell, tissue and animal, they have that length is equivalent at least or greater than the telomere of suitable contrast of age.These cells also have the gene expression pattern of young cell, as the EPC-1 activity that improves with respect to donorcells.And the present invention determines, opposite with nearest proposition produces clone's clone, and promptly " clone again " is fully feasible, and is unlimited multiple in theory, thus generation " super young " cell, tissue, organ and animal.

Think that at present telomere shortens the end of chromosome fracture that causes double-stranded undistinguishable, thereby send the signal (W.E.Wright and J.S.Shay 2000, Nat.Med.6 (8) 849-851) at the dna damage outpost of the tax office.

Yet, telomere may contain content more and more from telomere to centric degeneracy or non-telomere repetition DNA.

The appearance of these non-telomere repeat sequences causes the temporary transient dna damage outpost of the tax office.After repairing (as passing through exonuclease activity), cell can enter the cell cycle once more.Utilize subsequently nuclear transplantation to make the cell that causes death be grown to the aging in latter stage and cause that the even telomere repeat sequence that does not always exist in the nature extends the synthetic of array.

Contain and have these and extend and evenly the chromosomal cell of telomere repeat sequence and/or animal will be by rejuvenation, and have super young unique property, because there is less cell a cell time period in office at the dna damage outpost of the tax office in a large number.

The present invention is based on following discovery: nuclear transfer technology can be by activating the life-span of the active extension body cell of endogenous (cell) telomere (cell for example old and feeble or that stagnate near aging or the outpost of the tax office), by repairing the young pattern of series connection reiterated DNA sequences damage reinforcing gene expression.This with recently lose the method for proposition and compare for solving the telomere of finding in the animal that nuclear transplantation produces, have special advantage, its focus is the heterogenous expression of the telomerase gene of cloning, and shortens with the telomere that solves in the cloning mammal.

In this, the researchist of Geron company and Roslin institute cooperates recently, has merged the telomerase gene (hTERT) that Geron clones with nuclear transplantation, shortens in the hope of the telomere that solves among the clone.Referring to, for example, Bussiness Wire, on May 26th, 1999.This declaration is prior to the report of researchist on Nature of Roslin institute on the 27th in May, and this report claims to show also that with (how sharp after) other two sheep of nuclear transplantation clone telomere is shorter than suitable contrast of age.The researchist that Univ. of Massachusetts participates in clened cows also thinks, may have superiority on the life-span of cloned animal with the donorcells of external source telomerase gene transfection, although as if they find that nuclear transplantation makes the donorcells rejuvenation of aging.Referring to http://abcnews.go.com/sections/science/Dailv News/clones980522.html (1998).

The present invention is better than the method that proposes in order to express Telomerase by the telomerase gene of transfection because the activated end granzyme and in clone cell, tissue and animal the regeneration telomere length do not need genetic manipulation.In addition, the rise of the telomerase activation that reaches with the present invention is of short duration, when it is enough to prolong telomere length, can not give the infinite multiplication of composing type.Suppose and find that Telomerase composing type in the cancer cell of many types raises, and report delegation's moulding Telomerase Expression causes the rise of proto-oncogene c-MYC, then this advantage particularly important (article of D.Bead).Therefore, the gene that imports outside the Telomerase has also caused the possibility that inducing cell transforms, and might need to be intended to subsequently control the measure of being expressed Telomerase by rotaring redyeing gene.Therefore the active method of a kind of available cell self regulation mechanism control end granzyme preferably is used for inserting the external source copy of telomerase gene.

In addition, the present invention is better than the heterogenous expression of Telomerase, reaches the cell that prolongs the somatic cell culture generation a group rejuvenation of telomere subsequently with nuclear transplantation because cause telomere to shorten, and they all have consistent telomere and repeat proterties.Therefore, isolating individual cells has the big possibility that can strengthen propagation from this colony, and this colony will have than n cell has the outpost of the tax office still less to stagnate the peculiar property of cell, from but " super young ".

Therefore, the present invention includes with nuclear transplantation rejuvenation or raising normal somatic cell method of life.The somatocyte that has benefited from disclosed method comprises any somatocyte, for example owing to reach the restriction naturally of population doublings, or, cellular exposure maybe can be damaged under other condition of telomeric dna in hyperoxia pressure owing to harsh selection condition to complex inheritance change or condition, and near old and feeble cell.As mentioned above, this is particularly including the cell from patient with age related defects or illness or animal, as age relevant macular degeneration, immunosenescence neurodegenerative disease, as Parkinson's disease or Alzheimer, the diabetes of osteoarthritis, muscular dystrophy, skin aging, pulmonary emphysema, aneurysma, coronary heart disease, atherosclerosis, hypertension, cataract, the outbreak of growing up.The present invention also can be used for upgrading relevant illness with the cell that quickens, as muscular dystrophy, zoster, AIDS and liver cirrhosis.These methods are applicable to any purpose somatocyte and these cells purposes as the nuclear transplantation donor.

The somatic nuclear reprogramming that method of the present invention allows people to go down to posterity late period is an embryonism.Be many different cellular types by making the embryonic cell differentiation and growing, people can separate the purpose primary cell of rejuvenation or " youth " state again.And, because method of the present invention need prepare the embryonic stem cell that is divided into all different cellular types, thus the cell of any kind can be produced with any purpose primary cell, as long as somatic genome does not change down to influencing cell development.Therefore, the invention provides a kind of valuable method, in order to analyze the influence that waits identical hereditary change in the genetic background (being gene knockout or expression of heterologous genes) of external different cellular types.

For example, patient's somatic cell can be with nuclear transplantation correlation technique reprogramming, regenerate and revert to totipotency.Totipotent cell by these rejuvenation can obtain multipotential stem cell, as preceding matter, a matter, enangioblasts, hemopoietic stem cell.Can be transplanted in the donor with these pluripotent cells or by their deutero-cells, will be in patient's tissue " sowing " but healthy proliferative cell, as immunocyte, hemocyte, bone, muscle, nerve and other type.

Method of the present invention also by with described cell, they nuclear or karyomit(e) as the nuclear transplantation donor, improve the life-span of the cell (preferably mammalian cell more preferably is people's cell, for example needs the cell of rejuvenation) of hope.Preferably, this method can repeat, and cell, nuclear or the karyomit(e) self that obtains from the clone embryos that produces will be as the nuclear transplantation donor.In addition, donorcells is preferably genetically modified.

Method of the present invention by with described cell, by their deutero-nuclear or karyomit(e) as the nuclear transplantation donor or make the DNA of these cells be exposed to the embryonic cell type, further make the repetition DNA in the cell that people can recover to wish, and those participate in the aged gene to activate or regulate (reduce or strengthen and express), comprise the Telomerase in the cell of hope, for example need the mammalian cell of rejuvenation and the cell that stagnate at the outpost of the tax office.Such as hereinafter detailed description, this is a uneven discovery, because the present invention can provide a kind of method that participates in cell senescence and regulate the special molecular of cell survival of identifying.Particularly, the invention provides the test that evaluation can recover repetition DNA (as telomere), activates or suppress the compound (as Telomerase, gas, tPA etc.) of the gene that changes in the cell senescence process.

In view of the discovery that the inventor can be used to rejuvenation about nuclear transplantation or prolongs the life-span of mammalian cell (for example old and feeble or near old and feeble cell), no longer worry the hereditary age that clone's Mammals, tire, teratoma or embryo or inner cell mass or blastocyst have its parental generation.Therefore, the present invention also comprises with the nuclear transfer technology method of clone's Mammals, tire, teratoma or embryo etc. again.These again cloning process be particularly useful for producing the transgene mammal that the heterologous gene of expressing more than one or more than one genes are knocked out, because can produce these animals by clone technology, produce have identical genetic background the clone's and clone's Mammals again.These methods do not need mating or breeding, and they often produce other hereditary difference, thereby and can not obtain to contain on genome close linkage together the two of change gene of heredity knock out or the double transgenic Mammals.

The accompanying drawing summary

Fig. 1.The sign of nuclear transplantation donorcells cell aging.(A) cell is observed with phase microscope.Compare with former generation BFF cell (a), donorcells showed cell size and tenuigenin granularity increase (b).(B) the exemplary electronic Photomicrograph of BEF (a) and donor CL53 (b) cell.Note the nuclear (n) of the winding of CL53 cell.The CL53 cell is greater than the BEF cell, and their tenuigenin contains abundant lysosomes (arrow) and thick protofibril.Two pictures all are identical ratio of enlargement.Lines are represented 2 microns.Plastosome (in).(C) former generation (a, BEF) and (b, CL53) to enter DNA synthetic for cell, as by in 30 hours incubation processes evening generation ³The H-thymidine mixes mensuration (V.J.Cristofalo and B.B.Sharf (1973) Exp.Cell Res.76:419).Handle cell and carry out radioautograph, use microscopic examination then, and the nuclear of mark is marked.According at least 400 nuclears of scheme (Cristofalo and Sharf (1973)) counting of setting up, with the percentage ratio of the nuclear of determining mark.(D) by the Northern assay determination, donor CL53 cell shows that the EPC-1mRNA level reduces.Former generation (Y) and generation in evening (0) human fibroblasts (WI-38), former generation (Y have been marked; BEF) and evening generation (0; Donor CL53) ox inoblast and the isolating RNA of calf skin flbroblast strain from cloning.Converge growing into, and RNA (P.Chomczynski and N.Sacchi (1987) Anal.Biochem 162:156) is extracted in cessation of growth cessation from cell after 3 days in serum free medium.Equivalent RNA handles with oxalic dialdehyde, electrophoretic separation on sepharose, and electrophoretic transfer is to nitrocellulose filter, use standard conditions and total length EPC-1cDNA hybridization (D.G.Phinney, C.L.Keiper, M.K.Francis, K.Ryder (1994) Oncogene 9:2353).

Fig. 2.Normal cow by old and feeble somatic cell clone.(A) 5 months big CLS3-8, CL53-209, CL53-10, CL53-11 and CL53-12 (pet name is respectively Lilv, Daffodil, Crocus, Forsythia and Rose); (B) 10 months big CL53-1 (Persephone, insert).

Fig. 3.Nuclear transplantation recovers the ability in old and feeble donorcells propagation life-span.(A) originally the growth curve of BEF cell strain (*) with by evening for BEF cell (CL53 cell) clone's tire (ACT99-002) (o) growth curve of deutero-cell compare.(B) growth curve of CL53 donorcells shows to still have about 2 population doublings in the culture.(C) with the inoculation of clone's density, compare the multiplication capacity after 1 month for CL53 cell (n=97) evening.(D), show the ability of propagation expansion from the single cell clone of former generation BFF culture (originally) and former generation ACT99-002 (clone) with opposite by cell-derived clone of generation in evening.

Fig. 4.Telomere length is analyzed.(A) tool nuclear blood cell.In double blind experiment, pass through streaming fish analysis (N.Rufer, W.Dragowska, G.Thornbury, E.Roosnek, P.M.Lansdorp (1998) Nature Biotechnol.16:743) from the clone with peripheral blood sample control animal.With the double tool karyocyte sample (granulocyte of merging and lymphocyte) that obtains after the ammonium chloride infiltration splitting erythrocyte as stating by streaming fish analysis people (1999) J.Exp.Med.190:157 such as () N.Rufer.Deduct average background fluorescence by the mean fluorecence that obtains with FITC-mark telomere probe, calculate monocytic average telomere fluorescence.Note the relevant reduction of age of telomere fluorescent value in the normal cow, with relative long telomere in the cloned animal.(B) the segmental analysis of end limit.Isolating genomic dna the 7 all inoblasts of cloning tire (ACT99-002) cell age that obtain with old and feeble CL53 cell from control cells (transfection before BFF ox inoblast), old and feeble CL53 cell with by nuclear transplantation.The TRF of the dna fragmentation that obtains with HinfI/RsaI digestion back analyze as state on 0.5% sepharose, carry out 12 hours (telomere length is measured test kit, Pharmingen, San Diego, CA).Road 1: from CEPH lymphoblastoid human cell line 134105 contrast DNA; Road 2: biotinylated mark (Pharmingen); Road 3:TeloLow contrasts DNA (Pharmingen, average T RF length is 3.3kb); Road 4: old and feeble CL53 cell; Before the road 5:BFF inoblast transfection; Road 6:ACT99-002 (clone's) cell.(C) at the TRE analysis as B after 24 hours of electrophoresis on 0.5% sepharose, road 1:ACT99-002 cell (average T RF length is 19.3kb); Before the road 2:BFF056H inoblast transfection (average T RF length is 17.9kb); Road 3: old and feeble CL53 cell (average T RF length is 16.2kb); Road 4:TeloHigh control cells (Pharmingen, average T RF length is 11.3kb); Road 5: from CEPH lymphoblastoid human cell line 134105 contrast DNA; Road 6: the biotinylated λ DNA (molecular weight marker thing) that cuts with the HindIII enzyme.(D) to BIT ox inoblast, old and feeble CL53 cell and the fibroblastic streaming fish analysis of ACT99-002 before the transfection.With or not with FITC-3TA2) 3 peptide nucleic acid probes hybridization post analysis cell (being respectively grey and black histogram).Unicellular according to scattering of light character gate.Note higher autofluorescence as the old and feeble CL53 cell of nuclear donor.With linear level measurement fluorescence.Behind subtracting background fluorescence, ACT99-002 (clone's) cell has the highest fluorescence, follows by BFF (original) cell.As if old and feeble CL53 cell have minimum specific fluorescence.

Fig. 5.In the embryo who rebuilds rather than in donor ox inoblast, express Telomerase.Telomerase activation is measured test kit with telomeric repeat amplification method (TRAP), and (Pharmingen, SanDiego CA) measure.The lysate of the ox embryo (n=15) of adult donor aging (CL53) inoblast and reconstruction in the 7th day obtains in TRAP measures and uses.Road 1: from 4000 K562 human erythroleukemia cells' extract; Road 2:20bp mark; Road 3: cell-free extract; Road 4: heat treated embryo (n=1) extract; Road 5:n=10; Road 6:n=1; Road 7:n=0.1; Road 8:n=0.01; Road 9: from 4000 fibroblastic extracts of donor CL53; Road 10-11: inoblast extract contrast (not having TS template and heat-inactivated extract respectively); Road 12:20bp mark.All roads all contain internal control TRAP reactant (36bp).

Detailed Description Of The Invention

The present invention includes the method for rejuvenation normal somatic cell. " normally " body cell refers to that these cells that belong to body cell system are not that tumour produces or transform, be transplanted in the seedless egg mother cell or after otherwise contacting the factor that exists in the germ line cell, can be reprogrammed or be beneficial to embryonic development at the chromosome of the nuclear of this cell or this cell or this cell. Normal somatic cell can or genetic modification not. It is one of following at least that " rejuvenation ", the present invention refer to: described somatic possible Population multiplication; Other mark of EPC-1 activity or cell senescence reverses and is young state; Telomerase raises, and/or telomere increases. " super young " refers to that cell colony has the cell senescence sign than normal cell youth. " teratoma " refers to a component cell, contains the mesoderm, entoderm or the ectodermal derivative that are produced by totipotent cell.

In a preferred embodiment of the invention, will be used for normal somatic cell of the present invention is cell or the approaching old and feeble cell that stagnate at senile cell, the outpost of the tax office. Yet these methods are applicable to the normal somatic cell of any hope, preferably human cell. Copy aging and be a kind of physiological status (people (1989) Exp.Cell Res.184:138 such as West of the static undistinguishable that causes with density dependent inhibition by serum starvation or young cell growth; The people such as West (1996) Exp. Gerontol.31:175; With people (1998) Exp.Gerontol.33:67 such as Pignolo), as if comprise in the cell cycle near G₁The G on/S border₁The blocking-up in late period (Cristofalo and Pignolo (1996) Exp.Gerontol.31:111; Gorman and Cristofalo (1986) Exp.Cell Res. 167:87; Aging and the cytophylaxis mechanism with the people (1992) such as Cristofalo, the people such as Franceshi compile (NYAS, New York) 187-194 page or leaf).

Senile cell can be identified with the known several different methods in this area. For example, can copy old and feeble feature with the Ultrastructure analysis confirmation fibroblast that differs light microscope and electron microscope, comprise with young cell and comparing, obvious and active golgiosome, that cave in and the lobate nuclear that increases, large lysosome, and the increasing of cytoplasm microfilament (Lipetz and Cristofalo (1972) J. Ultrastruct.Res.39:43). In addition, the ability that senile cell enters the S phase reduces, and this passes through³The H thymidine mixes and reduces and senescence associated-β-galactosidase dyeing significantly increases definite people (1995) Proc.Natl.Acad.USA 92:9363 such as () G.P. Dimri. Senile cell shows that also compare EPC-1 (early stage population doubling level cDNA-1) people (1993) J.Biol.Chem.268:8949 such as () Pignolo mRNA level reduces with the former generation cell, compares gasI down regulation of gene expression people (1994) Exp.Cell Res.211:197-202 such as () Cowled with akinete.

Can come separating senescence cell senescent cells until reach irreversible growth retardation by propagated cell. For " approaching old and feeble ", the present inventor refers to that these cells have the ability that division is no more than about 3-6 time, and is preferably old and feeble less than 2 or 3 population doublings apart from copying. The normal somatic cell although produce to be used for the preferred meaning of the senile cell of nuclear transfer and to be going down to posterity is until finish more than about 90%-95% in life-span, and aging and Senescence state also can be by inducing the different reagent of cells contacting (comprising gene toxic agent and Cdk inhibitor) people (1998) Current Biol.8:351-354 such as () McConnell. The gene toxic agent induce be similar to old and feeble, be different from and be called as the static growth retardation that stagnate at the dna damage outpost of the tax office. In addition, approaching old and feeble cell also can obtain from (for example, have aging associated conditions) human or animal.

Method of the present invention can use the cell rejuvenation to produce clone's animal, perhaps can be used to for the normal purpose body cell of other purpose rejuvenation. These methods can comprise:

A. described body cell, somatic nuclear or somatic chromosome are transplanted in the suitable recipient cell of acceptor egg mother cell or ovum or other, to produce the embryo;

B. use this embryo to obtain to contain at least a kind of embryo, inner cell mass, blastodisc and/or stem cell of cell;

C. make embryo, inner cell mass, blastodisc and/or the Stem cell differentiation cell or tissue type for wishing;

D. separate the cell or tissue that obtains;

E. described cell or tissue is transplanted among the patient.

Even if cell teratoma, inner cell mass, blastodisc and/or the embryonic stem cell of the differentiation that separates according to the present invention will have no longer than donor body cell also the same with it at least long telomere, this also is one aspect of the present invention. The cell of these differentiation also should have youth or super young cell senescence sign. Also predicted a kind of method, the method cell or tissue, teratocarcinoma cell, inner cell mass, blastocyst cell or blastocyte conduct nuclear donor subsequently of differentiation. This method is specially adapted to separate normal somatic cell with multiple transgenosis or hereditary change, teratoma, ES cell etc., can infinitely repeat until reach the hereditary change of desired number.

The normal somatic cell that method of the present invention is used can be any cellular type. Suitable cell comprises immunocyte, such as B cell, T cell, dendritic cells, Skin Cell is such as the cell of horn cell, epithelial cell, cartilage cell, mound cell, nerve cell, core cell, esophagus cell, SF, Different Organs (comprising liver, stomach, intestines, lung, pancreas, cornea, skin, gall-bladder, ovary, testis, other reproductive organs, kidney etc.). Only cell generally is to breed easily the also cell of easily transfection in tissue is cultivated. Preferably, being used for the transfection allogeneic dna sequence DNA is fibroblast with the cellular type of carrying out nuclear transfer.

U.S. Patent number 5,945,577; The U.S. serial 08/888,057 of application on July 3rd, 1997; The U.S. serial 08/888,283 of application on July 3rd, 1997; The U.S. serial 08/935,052 of application on September 22nd, 1997; Disclose method and the scheme that realizes nuclear transfer with the U.S. serial 09/394,902 of application on September 13rd, 1999, all patents and application all are incorporated herein by reference.

Body cell can be from animal or the mammal of any type, and such as pig, goat, cat, dog, rat, mouse, ox, buffalo, sheep, horse, people, non-human primate, but ungulate cell preferably most preferably is the ox cell. Be used for the in the future source of self similarity of the egg mother cell of nuclear transfer or ovum, and can be identical or different kind with donorcells or DNA.

The animal of non-responsiveness can be to support the neoplastic any animal of monster, and non-responsiveness arrives the not degree of the teratoma repulsion of genesis and development. For example, the animal of non-responsiveness can be SCID or nude mice. In addition, cell also can be in the body differentiation or the fowl ovum in cell.

This method is particularly useful for separating the somatocyte with complexity or composition operation (being more than one transfection heterologous gene and/or gene knockout), may be difficult to somatocyte is kept looking being enough to influence the time of the hereditary change that is hopeful in cultivation.At first, somatocyte preferably becomes super young cell by nuclear transplantation, as the substrate of gene targeting.Therefore, can carry out the genetic manipulation first time, can after hereditary clock " resets ", carry out the genetic manipulation second time then according to method rejuvenation of the present invention then somatocyte.Therefore, according to the complicacy of genetic manipulation and the time span of rejuvenation process, the somatocyte of rejuvenation can have genomic at least one change according to the present invention.Also comprise the rejuvenation that produces with method of the present invention, genetically-altered cells.

The present invention comprises that also preparation and first kind of cell of different cellular types have the somatic method of homologous genes type.Rejuvenation method makes this method become possibility, its implementation is that chromosome transfer with the nuclear of first kind of somatocyte, first kind of primary cell or first kind of primary cell is in stoning acceptor ovocyte or other suitable recipient cell, perhaps make the protein in the somatocyte contact ovocyte, to produce teratoma or other noble cells group, wherein contain any derivative of ectoderm, mesoderm and entoderm.Also can use ovum in after fertilization stoning immediately.Therefore, in fact can separate the cell of any kind from teratoma, or by teratomatous cell development differentiation.The specific cell mark of specific purpose cellular type uniqueness is known in the art, can be used to identify clone's primary cell.

The dissimilar somatic method of cell for preparing and be used for nuclear transplantation generally comprises:

A. the nuclear of first kind of cell, each cell or the karyomit(e) of first kind of cell are transplanted in the suitable recipient cell of acceptor ovocyte or ovum or other, to produce the embryo;

B. use this embryo to obtain to contain at least a kind of embryo, inner cell mass, blastodisc and/or stem cell of cell;

C. inner cell mass, blastodisc and/or stem cell are expelled in the animal hemocyte culture or fowl ovum of non-responsiveness, form teratoma;

D. separate the teratoma that obtains;

E. in order to identify the specific cell type, separate different germinal layers;

F. separate and first kind of cell that cell is dissimilar.

In donorcells, nuclear or karyomit(e) embodiment from human body, can modify the genome of primary cell, make cell can not produce embryo alive.Its implementation comprises deactivation or knocks out one of one or more required genes of three kinds of germinal layers of formation, perhaps from growing " suicide " gene of regulating promoter expression specifically expressing the contained cellular type of non-purpose germinal layer.In addition, gene knockout or suicide gene are expressed the gene that can adhere to or grow special needs at the Mammals uterus.

As mentioned above, preferably, first kind of (nuclear donor) cell is a kind of inoblast.This method can constitute with any cell kind, can be used to produce clone organ and the tissue that is used to transplant in the human treatment clone especially.Therefore, this method cell of can choosing is implemented, and can produce a kind of tissue (being used for transplanting to the patient that needs are transplanted) with isolating primary cell.

The preferred primary cell type that produces by disclosed method is: neurone, bone sarcoplast, cardiac muscle, skin pancreatic beta cell, endotheliocyte, hematopoietic cell, skin cells, hair follicle cell, nephrocyte and neurocyte.This method further comprises from teratoma isolated cell and cultivate this cell in the presence of somatomedin, to promote further differentiation.Particularly, changed the genome of first kind of cell before nuclear transplantation, make the engineered tissue of new primary cell and generation express at least a treatment albumen, perhaps can not express may be to the deleterious natural protein of donor patient.Also comprise the cell and the tissue that produce by disclosed method.

The preferable use of the cell of disclosed method generation and tissue comprises noble cells type and the tissue that produces neurone, islet cells, liver cell, myocardial cell, hematopoietic cell and other hope from here.

These cells and tissue (randomly can be genetically modified) can be used for cell, tissue and organ transplantation, for example, the treatment of bum, hair transplantation, cancer, chronic pain, diabetes, nanism, epilepsy, heart trouble such as myocardial infarction, hemophilia, infertility, ephrosis, hepatopathy, osteoarthritis, osteoporosis, apoplexy, affective disorder, Alzheimer, enzyme defect, Huntington chorea, hypocholesterolemic, hypoparathyroidism, immune deficiency, Lou Gehrig disease, macular degeneration, multiple sclerosis, muscular dystrophy, Parkinson's disease, rheumatoid arthritis, Spinal injury and other wound.

Because nuclear transfer technology is useful in producing cloning mammal and clone cell and tissue, method of the present invention also can be used for producing the cloning mammal with complicated or compound hereditary change.In addition, the present invention also can be used for producing youth, super young animal more preferably.Particularly, the present invention includes a kind of method of cloning cloned animal again, wherein clone's animal is compared the cloned animal hereditary change again.This method was not attempted before discovery of the present invention, but this shows that nuclear transplantation rejuvenation evening is for cell and recover telomere length.If Ke Long Mammals has the identical hereditary age (that is, with first kind of hereditary age that nuclear donor is identical) with clone's hereditary animal again, according to clone's algebraically, the feasibility of this method will reduce.The result that the inventor obtains so far shows that this is not true, and the clone can arbitrarily carry out repeatedly in fact again, and will produce " super young " animal, embryo and cell.Super young animal generally has the enhanced immunity system and the enhanced top layer pigmentation that can produce antibody.

According to of the present invention a kind of preferably again cloning process comprise the following steps, and can be used to produce the cloned animal that has two genetic modifications at least:

A. from the purpose animal, obtain primary cell,

B. by inserting allogeneic dna sequence DNA and/or lacking n DNA primary cell is carried out the genetic modification first time,

C. the primary cell that uses the genetic modification first time is used for to enucleation oocyte or ovum or other suitable recipient cell nuclear transplantation as nuclear donor,

D. obtain to have clone's embryo, tire or the animal of first kind of genetic modification,

E. obtain clone's primary cell by clone's embryo, tire or animal,

F. by inserting allogeneic dna sequence DNA and/or lacking n DNA the primary cell of cloning is carried out the genetic modification second time,

G. use for the first time and for the second time clone's primary cell of genetic modification be used for to enucleation oocyte or ovum or other suitable recipient cell nuclear transplantation as nuclear donor,

H. obtain to have clone's embryo, tire or the animal again of first kind and second kind genetic modification.

This method can repeat arbitrarily repeatedly.Preferably, at least one is cloned step again and uses breeding for old and feeble or near the donorcells that stagnate at aging or the outpost of the tax office, makes that the telomere of clone's cell is regenerated after nuclear transplantation or recovered again.Particularly, method of the present invention further may further comprise the steps: embryo, tire or the animal cloned again of time cloning again, carry out genetic modification for the third time, and making again, the clone has first kind, second kind and the third genetic modification.Therefore, this method can be used to produce that a large amount of genes are knocked out, insertion or metathetical animal, and can be used to produce the animal that whole cell system was replaced or modified (being the immunity system that human immune system is replaced by ox), relates to complicated enzymatic pathway the gene substitution of (as the approach relevant with Rh factor, or complement cascade etc.).

Cloning process more of the present invention will produce complicated animal model, be used for research and several genes and/or cellular type diseases associated, perhaps can not be expressed the typical animal model replication that single transgene or purpose single-gene are knocked out.And, can be with the effect of therapeutic gene in these Research of Animal Model for Study complex inheritance backgrounds.Also can produce and detect and can regulate the different genes expressed products, knock out the gene that participates in causing immunne response with these animal models, with homology homologue displacement glue protogene or other structural protein gene, or the like.

The present invention includes wonderful discovery: senile cell can be by rejuvenation, EPC-1 active with can improve with aging relevant other cell marking, Telomerase can be activated, telomere can be extended, series connection repeats and can be repaired, and all these is by the nuclear transplantation method.Therefore, the present invention includes the discovery that activates telomerase activation and/or the active new way of EPC-1, its purposes prolongs telomere head and shoulders above and duplicates the life-span.We also predict other reparation to the series connection reiterated DNA sequences.Particularly, the invention provides that a kind of separating end granzyme activates, EPC-1 activates or the method for other aging genes involved activation mechanism, and with the machine-processed adjustable side granzyme of evaluation or the method for EPC-1 or other gene.

For example, can fractional separation the tenuigenin of ovocyte, it is combined with deadly cell or deadly nucleus or telomere, measure that Telomerase activates, EPC-1 activates (or with aging other relevant cell marking) and telomere prolongation.By this mensuration, can identify with separate ovocyte in be responsible for again the activated end granzyme, EPC-1 is active and/or the activeconstituents of other age relevant cell mark.Similarly, can be from ovocyte isolation of RNA or cDNA, and the transfection cell that causes death, or express being used for the active cell free system of detecting end granzyme, and the transfectional cell or the cell free system of telomerase activation can assay certificate be arranged.These methods can be in conjunction with the subtractive hybridization technology, to be enriched in embryo's forming process rather than the RNA that expresses in aging course.The gene that like this, can identification code may activate relevant enzyme with Telomerase.

Just at ovocyte or ovum participation Telomerase activated gene or the protein that may contain more than one of after fertilization in the stage.Do not wish to stick to any particular theory, the inventor thinks, grows in the ES cell that produces or the sexual cell at ovocyte or at ovocyte to have at least a adjusting albumen or RNA, and its participates in the adjusting of telomerase activation, with the ALT approach, can respond some aspect of senile cell environment especially.It is possible that direct activated end granzyme of these protein or RNA or telomerase gene are expressed, but these protein or RNA also are possible by suppressing telomerase inhibitor or aging or working near the ALT approach that exists in the senile cell or express.A kind of possible Telomerase activator is Oct4 or Rex.

For example, people such as Xu prove, the induced expression once more of retinoblastoma protein is old and feeble and suppress telomerase activation (Oncogene (1997) 15:2589-2596) in the tumour cell.Nearest report shows that also a gene on the karyomit(e) 3 may participate in the inhibition of transcribing of hTERT (catalytic subunit of Telomerase).Referring to http://claim.springer.de/EncRef/CancerResearch/samples/0001.htm.Also identified several can with the protein of Telomerase direct interaction, as p23/hsp90 (molecular chaperones) and TEP1 (Telomerase associated protein 1).The state-run breadboard researchist of LawrenceBerkeley declares to have cloned other two kinds of people's telomere associated protein (Tin1 and Tin2).Federal Technology Report, on December 30th, 1999, Partnership Digest, Technology Watch, the 9th page.Therefore, the regulation mechanism that present method is determined can work by the expression of combination or inhibition telomere enzyme binding protein or telomerase inhibitor, thereby improves telomerase activation, but also can or strengthen protein stability adjusting telomerase activation by the up-regulated gene expression.

The present invention includes at least a method that can directly or indirectly strengthen the gene of telomerase activation or ALT approach of identifying.These methods comprise at strengthening Telomerase or the active member of ALT in aging or the approaching old and feeble cell, cDNA or mRNA library that examination is produced by embryo or embryonic stem cell.These methods also can comprise to be identified and at least aly can directly or indirectly suppress Telomerase or the active gene of ALT, comprise, at the member that can suppress the telomerase activation in the embryonic stem cell, cDNA or mRNA library that examination is produced by old and feeble or approaching old and feeble cell.Telomerase activation can be measured by any known method of this area, comprises that measuring reporter gene expresses (as the hTRT gene) or fusion rotein.A kind of preferred reporter molecule is green fluorescent protein (GFP).Telomerase activation also can be used the TRAPeze test determination.Screening method can combine with other known method, to improve the usefulness of screening method, for example, if the test library is produced by ovocyte or ES cell, then before the examination of library, make cDNA or mRNA library and cDNA or mRNA library subtractive hybridization from senile cell, vice versa.

The present invention comprises that also evaluation can improve Telomerase, young gene expression pattern or the active method of protein of ALT, comprise: the cytoplasmic fraction of (a) collecting ovocyte, embryo or embryonic stem cell, (b) it is incorporated as in aging or the cell free system, contacts Telomerase, young genetic expression or the active change of ALT that special ovocyte or ES tenuigenin fraction cause with (c) mensuration near old and feeble cell design.Comprise that also screening can suppress the method for the compound of Telomerase or young genetic expression, comprise making contacting a kind of compound, to determine whether this compound suppresses Telomerase, young genetic expression or ALT activity with embryonic stem cell old and feeble or that produce near old and feeble donorcells by nuclear transfer technology.

The present invention comprises that also production is with young gene or regulate sequence (preferably being connected with suitable mark) cells transfected and can raise the method for the compound of young genetic expression with these cells evaluations.These sieve methods are identified adjustable ganglion cell's propagation or aged compound.Suppose that several genes may and work in the cycle in adjusting cell proliferation, comprise EPC-1, gas gene (people such as Ciccarelli, Mci.Cell.Bid.10 (4): 1525-1529 (1990), as gas-2 ,-3 ,-5 ,-7), PI-3 kinases (people such as Tresini, Cancer Res.58 (i): 1-4 (1998)), collagenase, tPA.

Another kind of screening is to modify the somatocyte that contains marker gene (being GFP), more preferably a kind of aging somatocyte, and wherein marker gene is the Telomerase fusion or combines with the gene that its expression changes with cell senescence.Telomerase gene and/or promotor can merge with the form of marker gene with a series of brachymemmas, then old and feeble the or approaching old and feeble cell of serviceable indicia construct transfection.Can utilize nuclear transplantation to identify zone or the gene promoter in the telomerase gene then or after nuclear transplantation, participate in activating the upstream of Telomerase Expression.

In addition, the present invention also comprises EPC-1 and other young gene is placed the control of allos (as adjustable, preferably strong) promotor down, and estimates to express the influence that strengthens or reduce telomerase activation and telomere.

The present invention comprise also that the somatocyte of genetic modification determines that telomere is regulated and the ALT approach in the situation of gene action.For example, when when in somatocyte, knocking out these genes before the ALT, can be according to the definite gene of the forfeiture of ALT function for ALT function key.

The present invention also comprises adjusting compound, protein and the nucleic acid of identifying by aforesaid method, with the medicinal compositions that contains them, they can be separated, and as according to external source Telomerase activator of the present invention be used for purposes described herein, that is, be used for the treatment of age-related disease, the treatment of aging tissue (as the retina cell), the usefulness of treatment for cancer and raising bone marrow transplantation.

By disclosed embodiment scope and spirit of the present invention have been described.

Embodiment 1---the tire donorcells

This tentative experiment shows, can recover the life-span of primary cultured cell with body-cell neucleus transplanting.When will from 6 age in week tire inoblast when being cultured to aging, they experience about 30 population doublings, the average cell cycle is 28-30 hour.For whether the nuclear transplantation of upchecking can save the no longer aging of these cells, with the tire that produces 40 days apart from the cell of old and feeble 0.8 population doublings.From 31 population doublings of the inoblast of this tire experience, by comparison, be 33 multiplications from the inoblast of the tire of the same age of normal pregnancy.Data show that nuclear transplantation can make the senile cell rejuvenation.

Embodiment 2

Clone calf by old and feeble donor somatocyte generation

A kind of somatocyte strain is from 45 days female ox tire (BFF), and with the selection box transfection of PGK guiding.Select cell 10 days with G418, isolate 5 neomycin resistance bacterium colonies, and pass through Southern engram analysis stable transfection with complete cDNA probes.Determine that by fish analysis a cell strain (CL53) always examines for 63%[] transgenic positive, select the nuclear transplantation research described in carry out this research.

Go down to posterity CL53 inoblast (it is characterized by the cytokeratin feminine gender, the vimentin positive) up to finishing more than 95% of its life-span.Utilize the cell of opticmicroscope to differ photo demonstration (Figure 1A), the morphology of cell is with consistent near the cell of life terminal point.More detailed analysis of Ultrastructure with electron microscope proves, the further feature of these cell displaying duplication agings comprises with young cell and compares outstanding and active golgi body that increases caves in and foliate nuclear, big lysosome and tenuigenin microfilament increase (Figure 1B) (27).In addition, show old and feeble phenotype for cell these evenings, shows that the ability that enters the S phase reduces, ³The H thymidine mixes minimizing (Fig. 1 C) and old and feeble relevant beta-galactosidase enzymes (SA-β-gal; Data not shown) remarkable increase (28) of dyeing have confirmed this point.And these cells are compared with former generation ox BEF cell and are shown that also EPC-1 (early stage population doubling level cDNA-1) (29) mRNA level reduces, and the reduction mode is similar to observed change in the WI-38 cell weathering process (Fig. 1 D).

(13) have rebuild 1896 bovine oocytes altogether by nuclear transplantation with old and feeble CL53 cell as mentioned above.Identify 87 blastocysts (5%) after cultivating for 1 week.Most of embryos (n=79) are transplanted in the synchronized acceptor of progestogen, and transplanting has 17 (53%) pregnancies in 32 acceptors of ultrasound examination after 40 days.The 7th all selectivity in gestation are taken out 1 tire ox (ACT99-002), and remaining 9 acceptors (29%) are still pregnant up to pregnant 12 weeks.3 in these cows in gestation (twin), the 253rd day and miscarriage in the 278th day in the 252nd day.Remaining 6 acceptors continue to grow to mature.The ratio that the blastocyst that uses old and feeble CL53 cell is formed (5%), early pregnancy (53%) and full-term pregnancy (19%) is compared with the contrast embryo (being respectively 5%, 45% and 13%) who uses non-old and feeble donor (CL57) the cell generation that is obtained by former generation BFF cell.

Calf CL53-1, CL53-8, CL53-9, CL53-10, CL53-1 1 and CL53-12 passed through elective caesarean section art childbirth (Fig. 2) on the the 280th, 273,273,273,266 and 266 day in gestation respectively.Genome analysis confirms to have transgenosis in two animals (CL53-1 and CL53-12) and in the tire ox of gestation selectivity taking-up in the 49th day.During birth, the performance of clened cows consistent with previously disclosed report (13,15,30,31).Baby weight (51.6 ± 3.6kg) general increasing, several calves have experienced pulmonary hypertension and respiratory distress at birth, and postvaccinal heating in 4th month.After preceding 24 hours, ox is energetic, does not have health problem.Yet we notice at preceding 2 has the diuresis/many drinks and the siccative of moderate to take in minimizing the middle of the month.Whether the existence that the generation of these complication and donorcells colony (strain isolated 53 or 57) and transgenosis are integrated all has nothing to do.After about 2 months, all calves all show well, are similar to by normal healthy controls calf in vitro fertilization and that the embryo transfer of body bone produces.All 6 cloned animals all survive after birth 5-10 is individual month and are normal.

Separate skin inoblast from the clone calf, mRNA is as preparation as described in Fig. 1 D.Cell expressing EPC-1 mRNA be on close level in or be higher than the former generation fetus cells.In order to get rid of the not possibility of senile cell generation cloned animal of sub-fraction, with normal and clone's density inoculation CL53 donorcells.Shown in Fig. 3 B, cell is apart from duplicating the population doublings that aging has 2.01 ± 0.11 (SEM).Experience population doublings more than 1 time or 2 times respectively with the cell less than 12% (11/97) and 3% (2/97) of clone's density inoculation, and do not have cell fission to surpass (Fig. 3 C) 3 times.On the contrary, 47.8 ± 0.9 population doublings of former generation (before the transfection) BFF cell experience, the interim average cell Cycle Length of logarithmic growth is 17.8 ± 0.7 (Fig. 3 A).

In order to check body-cell neucleus transplanting method whether can recover the propagation life-span of old and feeble donorcells, we have cultivated the inoblast of the 7 all tire oxen (ACT99-002) that come the self-selectively taking-up.Cell strain has experienced population doublings 85.3 ± 5.6 times, and the interim average cell Cycle Length of logarithmic growth is 17.7 ± 0.8 (Fig. 3 A).Single cell clone (n=5) is produced by clone (ACT99-002) tire suitable with primary (BFF) age, and separation is characterized by fibroblastic culture by immunohistochemical staining.These single cell clones are distinguished 31.2 ± 3.4 and 25.9 ± 2.9 population doublings (Fig. 3 D) apart from the clone's with the primary tire.These data show that the clone can restore the life-span of senile cell, and the cell age of tire is not reflected the number of times that the preceding donorcells of nuclear transplantation doubles in cultivation.

In order further to study the ability of nuclear transplantation rescue senile cell, with (CCCTAA) peptide nucleic acid probe (streaming FISH) in situ hybridization of direct FITC-mark after, with the flow cytometry control animal that the telomere length in the tool nuclear blood cell of cloned animal is suitable with the age (new calves (＜2 week age) and one's old mother ox (10-19 year)) compare (32,33).The result of two independent experiments (Fig. 4 A) shows, the contrast suitable with respect to the age, telomere length recovers (63.4 ± 1.7 to 51.0 ± 3.1 kMESF[mean value ± s.d. fully in the cloned animal, P＜0.0001, experiment 1], with 75.7 ± 1.7 to 61.4 ± 3.2 kMESF[P＜0.0001, experiment 2]).Really, the telomere of cloned animal is longer than 4 new calves (experiment 2) (75.3 ± 1.2 to 66.9 ± 1.4, P＜0.0002) statistically.For experiment 1 and experiment 2, the average telomere length of old ox is respectively 47.7 ± 0.7kMESF and 52.0 ± 3.6 kMESF.

Also utilizing the segmental Southern of end limit to analyze (34) has studied old and feeble (CL53), has contrasted the telomere length kinetics in (BFF before the transfection) and clone's (ACT99-002) cell.Result (Fig. 4 B-D) is consistent with the streaming fish analysis of tool nuclear blood cell.Be longer than old and feeble and former generation donorcells (be respectively 16.2 and 17.9kb) ( comparison road 4,5,6, Fig. 4 B) from the telomere (19.3kb) of cell of clone's embryo.Flow cytometry (streaming FISH, ref 32) to the same cell telomere length has confirmed these results (Fig. 4 D).Measure by TRAP, in rebuilding 7 days embryo, also detect high-caliber telomerase activation (Fig. 5, road 5-8), and the ox inoblast that is used as donorcells in the nuclear transplantation experiment is negative (Fig. 5, road 9).

Discuss

The telomere of not described in the past in the cloned animal recovers.Our result obviously is different from people's such as Sheils research (20), as if wherein the telomere in the sheep corrodes and can not repair after nuclear transplantation.Find 3 cloned animals---the telomere length control animal suitable with respect to the age of 6LL3 (how sharp, as to obtain from adult donor cells), 6LL6 (being produced by the embryo donorcells) and 6LL7 (being produced by the tire donorcells) reduces.The author proposes, because the participation of the gutless system of the generation of these animals, so the recovery fully of telomere length can not take place.They also propose, and it is consistent that donorcells is cultivated the time that spends before many sharp short TRF and the nuclear transplantation.As if these discoveries are significant, are not only because of the somatocyte by aging and produced the offspring who lives, and because the nuclear transplantation method has prolonged the telomere of animal, surpass the newborn control animal suitable with the age.Also do not know the life-span that whether can reflect these animals of measuring of telomere, than the cell that the former beginning and end operation tire by same age obtains arranged from the cell of clone's tire the longer propagation life-span although find.In fact, observed average T RF size meets these discoveries in the latter.

In discussion, usually ask the result whether animal that produces by nuclear transplantation is to use some rare cell rather than most of culturing cells about the clone.A large amount of cultures contain accessible different a plurality of pedigrees (43) of growing cells life-span.In fact, has the cell of maximum life the evening of using in this research at first for the cell representative.If still have 20 or the young cell subgroup of multi-population multiplication more in for culture in evening, their multiplication culture then are when seeing in the mouse cell culture of common spontaneous infinite multiplication.Anticipate this adverse opinion, we with donorcells with clone's density inoculation, and to the propagation life-span scoring of each cell.339 (98%) experience in 347 cells is less than 3 PD, and 347/347 (100%) experience 4 times or lower PD.And cell is cultivated in high serum (15%) concentration, and young cell is easy to fast breeding observe in plate.In the sample possibility of young cell thereby＜1/347.Still 7 animals (6 mature animals and 1 tire) from the fetus cells group of aging, have been cloned.Thereby be impossible (p＜0.001, card side) by undetectable young cell cloned animal accidentally.

People's such as this research and Shiels report (20) different may be owing to select difference on the donor somatocyte type.For example, people (12) such as Wilmut uses immobilized (G ₀) how sharp donor breast epithelial cell produce, and uses old and feeble (G in this experiment ₁) inoblast.In fact, nearest studies show that, the reconstruction of telomerase activation causes telomere to prolong and the fibroblastic infinite multiplication of normal people (35,36), and uses the prolongation of breast epithelial similar experiment not causing telomere and duplicate life-span prolongation (37).Propose explaining these differences (38) on the ability of Telomerase prolongation telomere or in the difference that adaptation is cultivated on the activated signal transduction path of back with cell.But, other investigator's reports, the heterogenous expression of hTERT can prolong telomere and make human breast epithelial cell immortalization (J.Shay, personal communications).

(39) are obviously raised in studies have shown that in cattle early embryo forming process Telomerase Activity in the past.The prolongation of telomere prompting in this research, the mechanism that the ox embryo of rebuilding by nuclear transplantation has telomere length regeneration and keeps is as long as karyomit(e) is stable before adhering to and in the growth whole process after adhering to.

Embodiment 3

Utilize the nuclear transplantation of adult donor cells

The above-mentioned data that obtain with tire inoblast donor are consistent with the experiment that the senile cell that obtains with adult animals carries out.Skin flbroblast is cultivated by 3 Holstein steer.Separate single cell clone, the counting population doublings is up to aging.Carry out nuclear transplantation with these old and feeble or approaching old and feeble inoblasts.The 6th week from the uterus, took out fetus in gestation, therefrom be separated into fibrocyte, cultivate up to aging.Cell is by immunohistochemical analysis, and demonstration is an inoblast.Adult animals when nuclear transplantation (counting is the PD number before old and feeble) and by its generation 6 age in week tire initiating cell in the number of times of population doublings in table 1, list.89.4 ± 0.9 PD are on average experienced in the isolated cells strain from clone's tire, by comparison, by normal age quite the cell strain that produces of the contrast tire in (6 age in week) be 60.5 ± 1.7 PD (P＜0.0001).These data show that the clone can restore (in fact prolonging) the somatic life-span, and the cell age of tire can not be reflected the number of times that the preceding donorcells of nuclear transplantation doubles in cultivation.

Table 1 is from fibroblastic population doublings of the tire of normal tire and the generation of adult senile cell clonal population

Clone's tire	The PD of original adult cell when nuclear transplantation	From the isolating fibroblastic PD of tire
			????25-1	????0.26	????90.14
????25-2	????0.0	????91.44
			????14-1	????4.0	????89.27
????14-2	????1.0	????90.34
			????22-1	????2.5	????85.86
Normal tire
			????1-1	????-	????59.64
????2-1	????-	????67.37
			????3-1	????-	????60.18
????3-2	????-	????59.82
			????3-3	????-	????55.66

Embodiment 4

The analysis of adult donor cells type

Biopsy obtains (obtaining) when butchering from all three germinal layers of the cow that grows up.Particularly collect following cell at least:

Ectoderm-keratinocyte

Mesoderm-skin flbroblast

Entoderm-enteric epithelium

Assess the part of above-mentioned three kinds of cellular types immediately, to determine telomere length.This can realize by different methods.The remainder of all 3 kinds of cellular types is cultivated up to aging.In culturing process, keep the also part of frozen each colony.According to the frozen different cell samples of special population doublings mark.

After this, estimate the telomere length of different cell samples, the cell that obtains when especially being included in aging.

Embodiment 5

Clone calf by the old and feeble donor somatocyte generation of growing up

To use the cell that obtains by embodiment 4 to obtain the clened cows tire.Particularly, with whole 3 kinds of cellular types, and the cell that uses different groups to double, promptly, produce Niu Kelong from the cell of old and feeble 0.8 population doublings.The clened cows tire is basically according to United States Patent (USP) 5,945, and 577 disclosed methods produce, and are incorporated herein by reference.In the time of 40 days, take out clone's tire, therefrom separate whole 3 types cells, for example, keratinocyte, skin hide fiber cell and intestinal epithelial cells.

In addition, also use two (40 days) of the same age wild tires to reclaim same 3 kinds of cells in contrast.These cells, and, be cultured to aging from clone's tire isolated cells.

In addition, after separating, measure the telomere length of these dissimilar culturing cells immediately from animal or from separate back these cells of refrigerated.Take out cell once more, and freezing up to aging from different cell colonys.Different cellular types to obtaining when different cell colonys double to the culturing cell from clone and wild-type embryo, are calculated telomere length afterwards.

The result of result and embodiment 4 relatively.These experiments are carried out at present.

Embodiment 6

EPC-1 in the young cell contrast old cell expresses

EPC-1 in relatively young or older people, ox cell, cloned animal and the contrast expresses.The result is as follows.

The result shows, and is younger than the young cell that obtains from intact animal from the young cell of cloned animal, measures as expressing by EPC-1.In these cells, also return to younger level as a kind of telomere length of mark.Its explanation may be, telomere has defects property when keeping length in immortality system, and telomere can not be often near internal sequence.Therefore, when from telomere when sequence is read in the kinetochore, (T ₂AG ₃) _nThe multiple fidelity destroys.This shows with figure below.

Old and feeble peripheral issue has used 3 ' 5 ' exonucleases to modify, and then exposure can recover TRF-2 bonded T ₂AG ₃Yet to a certain extent, damage has caused so-called cell aging in latter stage.

Wilmut thinks, is cloned in the animal and can be had problems by senile cell, shortens because telomere length has reflected the telomere of somatocyte donor nuclei.Yet our results suggest is in fact opposite.Culturing cell is to old and feeble or approaching old and feeble, or outpost of the tax office stagnation, makes cell loss T ₂AG ₃, eliminate less important damage with 3 ' 5 ' exonucleases, a chance can be provided, that is, this gene transplanting in enucleation oocyte or other embryonic cell, is followed outburst by telomerase activation, rebuild pure T ₂AG ₃Bundle (than the length of normal presence).These cells will have the longer life-span, and, because T ₂AG ₃Purity, in the interim cell cycle, seldom have cell to stagnate.This will cause being higher than normal mitotic cell index, comprise " younger " pattern that gene expands.

For more fully research, experimentize with suitable mammiferous culture of age with by young and senile cell and clone's sample of cell clone of containing or not containing the telomere of shortening.These cells grow to aging, and every 15pd is freezing.The cell aging mark that compares these cells.By the relatively genetic expression in these cells of known method (for example Northern trace or with suitable probe mark).

Embodiment 7

The levels of telomerase activity that raises in the embryo that nuclear transplantation produces

In order to study the mechanism that Telomerase extends, in the ox system, detected the telomerase activation level in the early embryonic development of nuclear transplantation (NT) back.The result (Betts and King, 1999, Dev.Genetics 25:397-403) about normal (IVF) ox embryo who announces before being similar to, Telomerase all is detectable in all stages of the early development of being analyzed.For NT and IVF contrast embryo, the telomerase activation level reduces during the age at the 8-16 cell, then in morula and blastocyst stage rising (data not shown).Yet the levels of telomerase activity in the NT blastocyst is higher 2 times than corresponding IVF blastocyst.

Embodiment 8

The contrast suitable with respect to the age, the super young immunologic function of cloned animal

In order to study the degree that immunosenescence reverses after the nuclear transplantation, and whether definite cloned animal has the contrast enhanced immunologic function more suitable than the age, after the different mitogens of external contact, detected from the cell of cloning cow with respect to immunne response from the cell that contrasts cow.To TSST (toxic shock syndrome, TSS toxin, but the bacterial superantigen of a kind of inducing T cell propagation) but reply and to the aspect of replying of the Phytolacca acinosa promitosis former (PWM) of inducing T cell and B cell proliferation, there were significant differences for cloned animal and control animal.All observed difference at the 2nd day and the 3rd day that cultivates.Replying of PHA (another kind of T cell mitogen is former) also observed difference, but change bigger.PHA is replied the observed significance of difference can be determined by detecting a large amount of experimenters.The difference of replying to ConA (a kind of T cell mitogen is former) is less, not remarkable statistically.For TSST and PWM, difference is about 2 times, and the TSST system showed 2.6 times influence in 72 hours.The result lists in following table.

Do not detect body internal reaction, suppose that observing cloned animal after inoculation has enhanced susceptibility mitogen.Yet, to the in vivo test of remembering antigenic skin delayed-type hypersensitivity lower danger will be arranged, and can be used for analyzing immunne response in the body.

Also can use the reagent that can be used for separating the specific cell type to carry out in vitro tests, to detect replying of specific cell type (being T cell subsets, B cell, scavenger cell etc.).Also available ordinary method detects the generation level of special division of cytoplasm.

The mean value of 78 hours cultures

Ox	Group	Non-	????Con_2	????Con_5	????PWM	????PHA	????TSST
								????C245	Contrast	????302	????39038	????35334	????4435	????3459	????4011
????C246	Contrast	????142	????27124	????28010	????7118	????1188	????6025
								????C247	Contrast	????327	????29154	????38478	????6555	????2373	????6945

????C248	Contrast	????512	????30374	????32072	????8046	????2972	????9421
								????C249	Contrast	????278	????56841	????49533	????11016	????11338	????15039
????C250	Contrast	????147	????29912	????24270	????11035	????2334	????13575
								Average SD		????285 ????137	????35407 ????11277	????34616 ????8889	????8034 ????2603	????3944 ????3701	????9169 ????4367
								????E1	?Exptl	????422	????64492	????61135	????16851	????12479	????22185
????E8	?Exptl	????234	????47037	????43113	????13496	????7986	????15199
								????E9	?Exptl	????472	????31735	????34609	????9511	????7130	????11320
????E10	?Exptl	????569	????43051	????36157	????14647	????10788	????23357
								????E11	?Exptl	????148	????49339	????41446	????21701	????2572	????44759
????E12	?Exptl	????352	????54187	????45248	????18829	????8795	????25907
								Average SD		????366 ????155	????48307 ????10968	????43618 ????9501	????15841 ????4273	????8292 ????3411	????23788 ????11630
The P-value		????0.032	????0.06	????0.09	????0.00	????0.05	????0.01
								Influence size:		????29％	????36％	????26％	????97％	????110％	????159％

Conclusion and extensive use of the present invention

Disclosed herein as us, according to Wilmut, the prolongation of telomere itself is unconspicuous in the somatocyte that nuclear transplantation causes.But even according to former result, it is unconspicuous beginning producing this fact of better result (longer telomere, more long-lived cell) from senile cell.

Although now, to slow down suggestion very inconsistent for telomere being shortened the mechanism of translating into the cell aging phenotype or the centre of cell cycle.Former article proposes, and the telomere multiple is lost the forfeiture with the key cells function of losing that causes the telomere gene gradually.Woodring E.Wright has proposed a kind of model several years ago, i.e. the heterochromatin relevant with telomere moved in telomere shortening, makes the telomere gene silencing, causes aging subsequently.Bryant Villeponteau has delivered almost opinion to the contrary,, has the taper heterochromatin relevant with telomere that is, and it shortens with telomere, and this activates the gene near telomere.Titia de Lange is proposing in the article about TRF2 and T ring recently, and senile cell no longer can and form the T ring in conjunction with TRF2.Yet another kind of possibility is, " scatters " in telomere and non-telomeric sequence, and more pure TTAGGG is wherein arranged at the telomere place, and is less in inside.When telomere in the somatocyte shortened, cell ran into the non-telomeric sequence at telomere place gradually, and they can not finally improve the level of activated p53 (p21 then) in conjunction with TRF2, cause slowing down of cell cycle and final stopping.Problem is that young cell propagation and aging suddenly may not be whole phenomenons or not have this phenomenon.May be the telomere of the damage that increases gradually of the quantity p21 that raises gradually.

Our results suggest, by old and feeble artificial removal's telomere, synthetic again fast correct TTAGGG after nuclear transplantation can be created under the young state than normal cell longer time proliferating cells and animal then.For evolution, selection will be than the longer cell of the survival of needs breedings and animal without any reason.Therefore, for kind of a system, the somatocyte that generation has than the TTAGGG of the more homogeneous of needs does not have reason.Culturing cell to old and feeble technology can be removed good and bad telomeric sequence effectively, and nuclear transplantation will give cell than the better life-span potential that normally has then.Even cell has the telomere length suitable with normal cell, this also is true.This will produce cell and the aging slow and longer animal of survival that has higher mitotic index for a long time.

Therefore, the nuclear transplantation method of using the telomere prolongation and even not having telomere to prolong can cause homogeneous TTAGGG new in the telomere synthetic again.Be not subjected to the restriction of hypothesis, inventors think that this can be by independent or take place with the rise of other gene (as cessation of growth cessation sequence (gas gene), collagenase, tPA etc.) bonded Telomerase, EPC-1.The cell that this will produce the longer and more healthy animal of survival and be used for human treatment " super young ".This is to prove for the first time promptly, to have a group cell and the tissue of overall phenotype by super young cell that promptly younger than normal blended a group young cell, that is, gene expression pattern and mitotic index are younger than normal young cell.The use side granzyme only prolongs the life-span of telomere and cell.Yet known to the inventor, all open reports all do not have evidence to show to obtain the cell of the younger or super youth of overall phenotype.In fact, many researchists report, slowed down old but also be not that old and feeble cell continues with Telomerase slowly but ad infinitum division.

A preferable use of the present invention is telomere to be short to reach the life-span of hope, but makes the homogeneity of TTAGGG the highest.This will optimize the delicate balance of life-span and cancer risk, that is, cell will not be the composing type infinite multiplication, and they consequently limit paracytic clone and expand not than essential longer telomere.

Estimate to have by senile cell clone's animal the character of uniqueness with this technology.For example, the animal of estimating to produce for fur will have the enhanced immunne response with more uniform hair color, will have stronger disease resistance, and have other advantage.

Said as us, concrete medical usage can be to be used for relevant disease of age, as age relevant macular degeneration, Parkinson's disease, Alzheimer, the diabetes of osteoarthritis, immunosenescence, skin aging, pulmonary emphysema, aneurysma, coronary heart disease, hypertension, cataract, the outbreak of growing up, or the like.In addition, also can be with cell renewal acceleration diseases associated such as muscular dystrophy, zoster, AIDS and liver cirrhosis by using the regenerated cell therapy.

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Claims (86)

1. the method for a rejuvenation primary cell comprises:

A. the nuclear of primary cell, primary cell or the karyomit(e) of primary cell are transplanted in acceptor ovocyte or the ovum, to produce the embryo;

B. use this embryo to obtain inner cell mass, blastodisc and/or stem cell;

C. inner cell mass, blastodisc and/or stem cell are expelled in the animal of non-responsiveness, form teratoma;

D. separate the teratoma that obtains;

E. in order to identify the specific cell type, separate different germinal layers;

F. separate cell with the primary cell same type.

2. the process of claim 1 wherein that described primary cell is a kind of senile cell or approaching old and feeble cell.

3. the process of claim 1 wherein that isolated cells contains and is not the average the same at least long telomere of the teratomatous cell of contrast of the same age that produces by nuclear transfer technology from nuclear transplantation teratoma.

4. the method for claim 3, wherein said telomere on average are longer than and are not the teratomatous cell of contrast of the same age that produces by nuclear transfer technology.

5. the method for claim 2, wherein said primary cell is a kind of inoblast.

6. the process of claim 1 wherein that described non-responsiveness animal is SCID or nude mice.

7. the process of claim 1 wherein that described primary cell has a change on the genome at least.

8. a generation and first kind of cell of different cellular types have the method for the primary cell of homologous genes type, comprising:

A. with the nuclear transplantation of first kind of cell in the acceptor ovocyte, to produce the embryo;

B. use this embryo to obtain inner cell mass, blastodisc and/or stem cell;

C. inner cell mass, blastodisc and/or stem cell are expelled in the animal of non-responsiveness, form teratoma;

D. separate the teratoma that obtains;

E. in order to identify the specific cell type, separate different germinal layers;

F. separate and first kind of cell that cell is dissimilar, wherein the telomere of new primary cell is the same long with the telomere that is not control cells of the same age in the teratoma that produces by nuclear transfer technology at least.

9. the method for claim 8, wherein said first kind of cell are a kind of senile cells or near old and feeble cell.

10. the method for claim 9, wherein said first kind of cell is a kind of inoblast.

11. the method for claim 8, wherein said primary cell are the types that is selected from unstriated muscle, skeletal muscle, cardiac muscle, skin and kidney.

12. the method for claim 8, dissimilar cells is cultivated in its existence that further is included in somatomedin down, to promote further differentiation.

13. the method for claim 11 wherein produces tissue (being used for transplanting to the patient that needs are transplanted) with described primary cell.

14. the method for claim 8, wherein the genome of first kind of cell changed before nuclear transplantation.

15. method isolated cells by claim 8.

16. the isolating tissue of method by claim 13.

17. the method for claim 7, wherein said hereditary change comprises the transfection of at least a heterologous gene.

18. the method for claim 7, wherein said hereditary change comprises the destruction of at least a natural gene.

19. the method for claim 14, wherein said hereditary change comprises the transfection of at least a heterologous gene.

20. the method for claim 14, wherein said hereditary change comprises the destruction of at least a natural gene.

21. one kind is carried out the method for compound genetic manipulation to primary cell, is included in and utilizes this primary cell of nuclear transplantation rejuvenation in the acceptor ovocyte between the genetic manipulation, wherein before nuclear transplantation be old and feeble or near the state of aging with this passage.

22. one kind is carried out the method for compound genetic manipulation to primary cell, is included in and utilizes this primary cell of nuclear transplantation rejuvenation in the acceptor ovocyte between the genetic manipulation, wherein before nuclear transplantation is old and feeble sample or near old and feeble sample state with this cell induction.

23. the method for claim 21, rejuvenation produce average the same with the contrast embryonic cell of the same age long embryonic cell of the telomere that contains thus.

24. a kind of primary cell according to the method hereditary change of claim 21.

25. the cell of production and claim 24 has the method for animal of the hereditary change of homologous genes type, comprising:

A. with the nuclear transplantation of described cell in the acceptor ovocyte,

B. produce embryo or embryonic stem cell with this tool nuclear ovocyte,

C. embryo or embryonic stem cell are imported in the female receptor and

Embryo or embryonic stem cell are grown fully, make female childbirth and primary cell that the new born animal of homologous genes type be arranged.

26. the animal of the hereditary change that the method by claim 25 produces, this animal contain average the same with control animal of the same age at least long telomere.

27. a method of cloning a kind of cloned animal again with nuclear transfer technology, wherein being used for supplying again the donorcells of clone's nuclear is a kind of aging or approaching old and feeble cell.

28. the method for claim 25, wherein clone's animal is compared hereditary change with cloned animal again.

29. a production contains the method for clone's inner cell mass, blastocyst, teratoma, embryo, tire or animal again of two kinds of genetic modifications at least, comprising:

A. from the purpose animal, obtain primary cell,

B. by inserting allogeneic dna sequence DNA and/or lacking n DNA primary cell is carried out the genetic modification first time,

C. make the primary cell propagation of genetic modification be old and feeble or approaching aging,

D. the cell that uses the old and feeble or approaching aging of genetic modification for the first time is used for to enucleation oocyte or the nuclear transplantation of stoning zygote as nuclear donor,

E. obtain to have clone's inner cell mass, blastocyst, teratoma, embryo, tire or the animal of first kind of genetic modification,

F. obtain clone's primary cell by clone's inner cell mass, blastocyst, teratoma, embryo, tire or animal,

G. by inserting allogeneic dna sequence DNA and/or lacking n DNA the primary cell of cloning is carried out the genetic modification second time,

H. make the primary cell propagation of cloning for the second time up to old and feeble or approaching old and feeble,

I. use have for the first time and for the second time genetic modification old and feeble or near old and feeble clone's primary cell as nuclear donor, be used for to enucleation oocyte or the nuclear transplantation of stoning zygote and

J. obtain to have clone's inner cell mass, blastocyst, teratoma, embryo, tire or the animal again of the first time and the genetic modification second time.

30. the method for claim 29, it further comprises the step that inner cell mass, blastocyst, teratoma, embryo, tire or the animal cloned are cloned again again, wherein carry out genetic modification for the third time, make that the clone has the first time, the second time and genetic modification for the third time again.

31. the method for claim 30 wherein produces clone again by nuclear transfer technology with old and feeble or approaching old and feeble donorcells.

32. the method for claim 29, wherein said clone again contains at least and is not the average the same long telomere of control animal of the same age that produces with nuclear transfer technology.

33. the method for claim 31, wherein said clone again contains at least and is not the average the same long telomere of control animal of the same age that produces with nuclear transfer technology.

34. the method for claim 29, wherein genetic modification relates to the gene of being responsible for immunologic function.

35. the method for claim 29, wherein the purpose animal is a kind of ungulate.

36. the method for claim 35, wherein the purpose animal is an ox.

37. one kind is restored that aging, the outpost of the tax office are stagnated or near the method for old and feeble cell survival, comprises that nuclear transplantation with this cell is in the acceptor ovocyte.

38. the method for claim 37, wherein the acceptor ovocyte is not of the same race with old and feeble or approaching old and feeble cell.

39. the method for claim 37, it further comprises by described tool nuclear ovocyte generation embryo or embryonic stem cell.

40. identify at least a method that can directly or indirectly improve the gene of telomerase activation for one kind, comprise at improving aging or near the member of senile cell Telomerase Activity, cDNA or mRNA library that examination is produced by embryo or embryonic stem cell.

41. the method for claim 40 is expressed the raising of measuring telomerase activation by measuring enhanced Telomerase reporter gene.

42. the method for claim 41 wherein makes up described Telomerase reporter gene, makes it to contain the hTRT gene that merges with a kind of reporter gene.

43. the method for claim 42, wherein this construct contains gene fusion.

44. the method for claim 42, wherein this construct contains the protein fusion.

45. the method for claim 40 is by the telomerase activation of TRAPeze test determination raising.

46. the method for claim 40 before the examination of library, makes described cDNA or mRNA library and carries out subtractive hybridization from the cDNA or the mRNA library of senile cell.

47. identify at least a method that can directly or indirectly suppress the gene of telomerase activation for one kind, comprise at the member that can suppress the embryonic stem cell Telomerase Activity cDNA or mRNA library that examination is produced by old and feeble or approaching old and feeble cell.

48. the method for claim 47 is expressed the reduction of measuring telomerase activation by measuring the Telomerase reporter gene that reduces.

49. the method for claim 47 wherein makes up described Telomerase reporter gene, makes it to contain the hTRT gene that merges with a kind of reporter gene.

50. the method for claim 49, wherein this construct contains gene fusion.

51. the method for claim 49, wherein this construct contains the protein fusion.

52. the method for claim 47, telomerase activation reduces by protein interaction, and the reduction of telomerase activation is by the TRAPeze test determination.

53. the method for claim 47 before the examination of library, makes described cDNA or mRNA library and carries out subtractive hybridization from the cDNA or the mRNA library of embryonic stem cell.

54. an evaluation can improve the method for protein of EPC-1 and/or telomerase activation, comprising:

A. collect the cytoplasmic fraction of ovocyte,

B. it is incorporated as aging or near in the cell free system of old and feeble cell design and

C. measure because Telomerase and/or the EPC-1 activity change that the special ovocyte tenuigenin fraction of contact causes.

55. method genes identified by claim 40.

56. method genes identified by claim 47.

57. protein of identifying by the method for claim 54.

58. a screening can suppress the method for Telomerase and/or the active compound of EPC-1, comprise making contacting a kind of compound, to determine whether this compound suppresses Telomerase and/or EPC-1 activity with embryonic stem cell old and feeble or that produce near old and feeble donorcells by nuclear transfer technology.

59. method compounds identified by claim 58.

60. a gene that contains claim 55, or the medicinal compositions of its part or transcription product, purpose are to improve the telomerase activation that needs to improve active patient.

61. the medicinal compositions of the gene product of a genes encoding that contains claim 55, purpose are to improve the telomerase activation that needs to improve active patient.

62. a gene that contains claim 56, or the medicinal compositions of its part or transcription product, purpose are to suppress the telomerase activation that needs suppress active patient.

63. the medicinal compositions of the gene product of a genes encoding that contains claim 56, purpose are to suppress the telomerase activation that needs suppress active patient.

64. a proteinic medicinal compositions that contains claim 58, purpose are to improve the telomerase activation that needs to improve active patient.

65. the proteinic gene of the claim 58 of encoding.

66. a medicinal compositions that contains the gene of claim 65, purpose are to improve the telomerase activation that needs to improve active patient.

67. a medicinal compositions that contains the compound of claim 59, purpose are to suppress the telomerase activation that needs reduce active patient.

68. method that activates endogenous Telomerase and/or EPC-1 for the life-span that prolongs primary cell.

69. the cell with multiplication capacity of rejuvenation, it is to produce by the compound that makes cell with somatocyte life-span or its DNA contact sexual cell or embryonic cell or its fractional separation.

70. the cell of claim 69, wherein the homotype that this cell is suitable with respect to age somatocyte of the same race has the EPC-1 activity of raising and/or the telomere that prolongs.

71. the cell of claim 69, it is selected from: people, ox, horse, dog, cat, pig, mouse, rat, goat, sheep, cavy, bear, rabbit.

72. the cell of claim 69, it is a kind of human cell.

73. contain the DNA of the prolongation telomere that the cell of with good grounds claim 60 produces.

74. the DNA of claim 73, it derives from human body cell.

75. a generation has the method for cell of the multiplication capacity of rejuvenation, method is to make the cell that belongs to somatocyte system or its DNA contact ovum, ovocyte, embryonic cell or the component of fractional separation therefrom.

76. the method for claim 75, wherein said somatocyte are old and feeble, approaching aging or stagnate at the outpost of the tax office.

77. the method for claim 75, wherein said somatocyte are a kind of human cells.

78. the method for claim 77, wherein said somatocyte obtains from suffering from old and feeble associated conditions or more increasing newly the human body of relevant illness with cell.

79. the method for claim 78, wherein said illness is selected from: AIDS, muscular dystrophy, neurodegenerative disease, hypertension, immune deficiency, osteoarthritis and diabetes.

80. by clone's non-human embryo, zooblast or non-human animal that nuclear transplantation produces, wherein donorcells or nuclear are the cells that stagnate at a kind of senile cell or the outpost of the tax office.

81. improved nuclear transplantation method, it can produce, and the telomere prolongation is compared in the contrast suitable with the age and/or EPC-1 is active improves, and/or telomerase activation improves, and/or multiplication capacity strengthens or the life-span prolongs non-human embryo or animal or human's class or non-human cell, wherein this improvement comprise use a kind of aging, near the donorcells old and feeble, that stagnate at the outpost of the tax office or DNA as donorcells or DNA.

82. an evaluation can influence the method for the compound of cell senescence or aging, comprising:

(i) produce a kind of EPC-1 gene or EPC-1 and regulate the sequence cells transfected; With

This regulates the compound of sequence (ii) to identify " unlatching ".

83. the method for claim 82, wherein said EPC-1 regulates sequence or gene effectively is connected with a kind of DNA, and its expression is detectable.

84. an eukaryotic cell, its is by the EPC-1 gene or the transfection of associated adjustment sequence that effectively are connected with marker DNA.

85. an eukaryotic cell, it maybe can be regulated the EPC-1 gene transfection that strong promoter effectively is connected with composing type.

86. the cell of claim 85, wherein said EPC-1 gene are regulated sequence with CMV, PGK or other non-EPC-1 and effectively are connected.

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