METHODS AND COMPOSITIONS FOR ENHANCING DEVELOPMENTAL POTENTIAL OF OOCYTES AND ZYGOTES

Abstract: The invention relates to compositions and methods for enhancing the developmental potential of oocytes or zygotes by increasing intracellular levels of replicative mitochondria in the oocytes or zygotes. In one aspect of the invention, the intracellular levels of replicative mitochondria are increased by introducing replicative mitochondria into the oocytes or zygotes. The oocytes may be fertilized to obtain a zygote with increased intracellular levels of replicative mitochondria. The methods and compositions may be used to improve in vitro fertilization and embryo transfer methods, and nuclear transfer techniques.

Description: TITLE: Methods and Compositions for Enhancing Developmental Potential of Oocytes and Zygotes FIELD OF THE INVENTION The invention relates to compositions and methods for enhancing the developmental potential of oocytes, zygotes, and preimplantation embryos. BACKGROUND OF THE INVENTION With in vitro fertilization (IVF) and other assisted reproductive technologies, about 50% of human embryos undergo a suicide program of active cell death and become fragmented. Some infertility patients produce only fragmented embryos, which appears to be the cause of their failure to conceive or carry a pregnancy to term. In all mammals, including humans, zygote development and the first cleavage divisions depend upon maternal RNA and protein products accumulated during oogenesis. Reproductive failure can be attributed to the lack of cleavage in the developing embryo. This phenomenon can be traced to a defect in the composition of the oocyte cytoplasm. Maternal cytoplasmic components are involved in embryonic arrest, because the "2-cell block" in mice can be overcome by transplantation of ooplasm from zygotes of non-arresting strains into the zygotes of arresting strains (Muggleton-Harns et al. Nature. 1982 Sep 30;299 (5882):460-2). Similar oocyte cytoplasm transfer experiments demonstrated improvement in the developmental capacity of immature eggs in mice (Flood et al, 1990). Later, Cohen and colleagues obtained a pregnancy in a patient with a history of consistently fragmented embryos by transfer of donor oocyte cytoplasm into the patient's oocytes at the time of lntracytoplasmic sperm injection (Cohen et al, Lancet, 1997 Jul 19;350(9072): 186-7). More recently, Lanzendorf et al (Fertil Steal. 1999 Mar;71(3):575-7) demonstrated that frozen-thawed oocyte cytoplasm microinjected into oocytes, improved their developmental competency after fertilization, and resulted in a twin pregnancy in a patient who previously produced only fragmented embryos Nuclear transfer as a means of producing identical individuals (clones) has been successfully performed in several mammalian species including goat, sheep, pig, cattle, and mice. In all of these cases, efficiency of this technique is very low. While development of reconstituted embryos to the blastocyst stage is moderate (-40%, Ogura et al, 2000 Biol. Reprod. 62(6): 1579-84, 2000; Mol. Reprod. Development 57:55-59, 2000), live birth rate is unexpectedly low (1-7%, Ogura et al, 2000, supra, Polejaeva et al Nature 2000 Sep 7;407(6800):86-90). Moreover, extensive fetal and early neonatal death has previously been reported m offspring obtained by nuclear transfer (Rideout WM 3rd, Wakayama T, Wutz A, Eggan K et al 2000 Nat Genet Feb;24(2):109-10). Thus, there is a need to enhance the developmental potential of oocytes to improve reproductive technologies including nuclear transfer methods. SUMMARY The invention relates to a method for enhancing developmental potential of oocytes comprising increasing intracellular levels of rephcative mitochondria in the oocytes. In an embodiment of the invention, the intracellular levels of rephcative mitochondria are increased by introducing rephcative mitochondria into the oocytes. A method of the invention may additionally compπse fertilizing the oocytes to obtain a zygote with mcreased intracellular levels of rephcative mitochondπa. The invention also relates to a method for enhancing developmental potential of zygotes compπsing increasing intracellular levels of rephcative mitochondπa in the zygotes. In an embodiment of the invention, the intracellular levels of rephcative mitochondπa are increased by introducing rephcative mitochondria into zygotes. The invention further relates to an oocyte or a zygote with increased intracellular levels of rephcative mitochondria obtamed from a method of the invention. In a further aspect the invention relates to a composition compπsing rephcative mitochondπa for enhancing developmental potential of oocytes or zygotes, and for treating and preventing heπtable mitochondπal diseases. The composition may compπse cryopreserved mitochondna. In another aspect, the invention provides a method for fertilizing oocytes compπsing removing oocytes from a follicle of an ovary, introducing rephcative mitochrondπa into the oocytes, and fertilizing the resulting oocytes with spermatozoa. In a still further aspect the invention provides a method for stonng and then enhancing the developmental potential of oocytes compπsing cryopreservmg immature oocytes, thawing the cryopreserved oocytes, and introducing rephcative mitochondπa into the oocytes. A method is also contemplated for enhancing the developmental potential of oocytes compπsing cryopreservmg rephcative mitochondπa, thawing the mitochondπa, and introducing the rephcative mitochondπa into oocytes. The methods and compositions of the invention improve the quality of the oocytes that are being fertilized and the quality of zygotes, to increase the rate of success m embryo development and ongoing pregnancy The methods and compositions are particularly useful m enhancing the developmental potential of oocytes or zygotes with mitochondπal DNA mutations or abnormal mitochondπal metabolic activity. In an aspect, the invention provides a method for improving embryo development after in vitro fertilization or embryo transfer in a female mammal compπsmg implanting into the female mammal an embryo deπved from an ooctye or zygote containing mcreased intracellular levels of rephcative mitochondπa. The invention also provides a method for reducmg the detrimental effects of mitochondπal DNA mutations (e g. deletion or rmssense mutations) m the progeny of an individual affected by such mutations compπsmg introducing mto oocytes or zygotes from the individual rephcative mitochondna that does not contain the DNA mutations (i.e. healthy mitochondπa). The invention further provides an oocyte or a zygote compπsing both mitochondπa with mitochondπal DNA mutations, and punfied and isolated rephcative mitochondna that do not contain the mitochondπal DNA mutations (i.e. healthy mitochondπa). The mvention also relates to a method for treating heπtable mitochondπal diseases m the progeny of an individual affected by such diseases compnsmg mtroducmg mto oocytes or zygotes from the individual rephcative mitochondπa compπsmg mitochondπa that does not contain the DNA mutations (i.e. healthy mitochondπa). In an aspect of the mvention, the oocyte is a recipient ooctye m a nuclear transfer method. Thus, the mvention relates to a method for enhaincing developmental potential of recipient oocytes in a nuclear transfer method compnsmg mtroducmg rephcative mitochondna mto the recipient oocytes. The mvention also contemplates recipient oocytes compπsmg rep cattve mitochondna, and blastocyts, embryos, and non-human animals formed from the nuclear transfer methods of the invention. In conventional nuclear transfer methods, the donor nucleus is placed in an enucleated oocyte obtamed from a different mdividual. Thus, mitochondria in the recipient oocyte have not-co-existed with the donor nucleus. Since mitochondria are always maternally inheπted, their replication, transcπption, translation, and function does not only depend on mitochondπal DNA, but is tightly intercalated with the nuclear genome that co-exists with the mitochrondna. The invention by introducing rephcative mitochondna mto recipient oocytes enhances the developmental potential of the recipient oocytes. This is expected to increase the live birth rate in nuclear transfer methods. In an embodiment, the invention provides a method of cloning a non-human mammalian embryo by nuclear transfer compπsmg (a) introducing a donor cell nucleus deπved from a donor cell of a non-human mammal, and rephcative mitochondπa preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the non-human mammal from which the donor cell nucleus is deπved, into an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) cultuπng the nuclear transfer unit to form an embryo The method may further compnse permitting the embryo to develop mto a cloned mammal. Therefore, the invention also provides a method of cloning a non-human mammal by nuclear transfer compπsing (a) introducing a donor cell nucleus denved from a donor cell of a non-human mammal, and rephcative mitochondπa preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the non-human mammal from which the donor cell nucleus is denved, mto a non-human mammalian enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) cultunng the nuclear transfer unit to form an embryo; (c) implanting the embryo into the uterus of a suπogate mother of said species, and (d) permitting the embryo to develop mto the cloned mammal. In yet another embodiment, a method of clonmg a non-human mammalian fetus by nuclear transfer is provided compnsmg the following steps: (a) mtroducmg a donor cell nucleus from a donor cell of a non-human mammal, and rephcative mitochondria preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the non-human mammal from which the donor cell nucleus is denved, mto an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) culturmg the nuclear transfer unit until greater than the 2-cell developmental stage; and (c) transfemng the cultured nuclear transfer unit to a host non-human mammal of the same species such that the nuclear transfer unit develops into a fetus. The method may also compnse developing the fetus into an offspring. In a further aspect the invention provides a recipient oocyte compnsmg a penvitelline space and a donor cell nucleus and rephcative mitochondria preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same individual from which the donor cell nucleus is dervied, deposited in the penvitelline space Other objects, features and advantages of the present invention will become apparent from the following detailed descπption. It should be understood, however, that the detailed descπption and the specific examples while indicating prefeπed embodiments of the invention are given by way of illustration only, since vaπous changes and modifications within the spiπt and scope of the invention will become apparent to those skilled in the art from this detailed descnption BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar graph showing the effect of mitochondπa injection on preimplantaion embryo development DETAILED DESCRIPTION OF THE INVENTION The term "oocytes" refers to the gamete from the follicle of a female animal, whether vertebrate or invertebrate. Preferably, the animal is a mammal, and more preferably is a non-human pπmate, a bovme, equine, porcme, ovme, caprine, buffalo, guinea pig, hamster, rabbit, mice, rat, dog, cat, or a human Suitable oocytes for use in the invention include immature oocytes, and mature oocytes from ovanes stimulated by administering to the oocyte donor, in vitro or in vivo, a fertility agent or fertility enhancmg agent (e g. inhibin, inhibin and activin, cloπuphene citrate, human menopausal gonadotropms including FSH, or a mixture of FSH and LH, and/or human choπomc gonadotropms) In some embodiments of the invention, the oocytes are aged (e.g. from humans 40 years +, or from animals past their reproductive prime). The oocytes m some embodiments of the invention contain mitochondπal DNA mutations. Methods for isolating oocytes are known in the art. In the nuclear transfer embodiments of the mvention oocytes are used as recipient cells (such cells are referred to herein as "recipient oocytes") The recipient ooctyes are obtamed from non-human mammals, in particular domestic, sports, zoo, and pet animals mcludmg but not limited to bovme, ovine, porcine, equme, capnne, buffalo, and gumea pigs, rabbits, mice, hamsters, rats, primates, etc. The term "zygote" refers to a fertilized oocyte pπor to the first cleavage division. The expression "enhancing the developmental potential of oocytes" refers to increasing the quality of the oocyte so that it will be more capable of bemg fertilized and/or enhancmg mitochondπal function or activity m the oocyte for subsequent development and reproduction. Increasmg the quality of the oocyte, and thus the fertilized oocyte (e.g. zygote), preferably results m enhanced development of the oocyte into an embryo and its ability to be implanted and form a healthy pregnancy. The expression "enhancing the developmental potential of zygotes" refers to increasing the quality of the zygotes and/or enhancmg rmtochondnal function or activity m the zygotes for subsequent development and reproduction. Increasing the quality of the zygotes, preferably results in enhanced development of the zygotes into an embryo and their ability to be implanted and form a healthy pregnancy. Quality can be assessed by the appearance of the developing embryo by visual means and by the IVF or nuclear transfer success rate. Cntena to judge quality of the developing embryo by visual means include, for example, their shape, rate of cell division, fragmentation, appearance of cytoplasm, and other means recognized in the art of IVF and nuclear transfer. "Spermatozoa" refers to male gametes that can be used to fertilize oocytes. "Heritable mitochondπal diseases" refers to diseases caused by defects in mitochondπal DNA or by defects in nuclear genes that are important to mitochondnal function. Examples of rmtochondnal diseases include but are not limited to Kearns-Sayre syndrome, MERRF syndrome (Myoclonic Epilepsy with Ragged Red Fibres), MELAS syndrome (Mitochondnal Encephalopathy, Myopathy, Lactic Acidosis and Stroke-like episodes), and Leber's disease (I. Nonaka, Cuπent Opinion in Neurology and Neurosurgery, 5 (1992) 622) The term "rephcative microchondna" refers to a preparation of punfied mitochondna that are capable of replicating during embryo development and increasing mitochondnal copy number or function. The rephcative mitochondna is substantially free of other cytoplasmic components mcludmg nuclear DNA, mRNA, protems, antioxidants, and organelles other than mitochondπa. The rephcative mitochondria preparations are at least 60% free, preferably 75% free, and most preferably 90% free from other cytoplasmic components. Preferably the rephcative mitochondπa preparations contam greater than 70%, more preferably greater than 80%, most preferably greater than 90% functional mitochondria. A rephcative mitochondna preparation typically contams about 2,000 to 20,000 mitochondna in a volume of 5 to 15 picoL. For the non-nuclear-transfer embodiments of the invention, rephcative mitochondna are preferably denved from any stem cell (e.g. hematopoietic, embryonic, trophoblastic, primordial germ cells) or from any immortalized cell lme (e.g. cancer, or intentionally transformed somatic cells) of any species, preferably human. The cells are preferably free of the common mitochondnal deletion mutation found clinically m patients with KSS syndrome (i.e. deleted 4799bp region at nt 8470-13,447; see Simonnetti et al, 1992) and any other pathologic mitochondπal DNA mutation. Particular methods for preparing rephcative mitochondna are illustrated herem and are descπbed in Darley-Usmer VM., Rickwood D, Willson MT. Mitochondna, a Practical Approach, Oxford Washmgton DC, IRL Press, 1987, pp. 1-16. Stem cells used to prepare the rephcative mitochondπa can be genetically modified by genetic engineering techniques. A transgene may be introduced mto the cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, hpofection, electroporation, or micromjection. Suitable methods for transforming and transfecting cells can be found m Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. (See also Nolta et al Blood. 1995 Jul 1 ,86(1):101-10; and Nolta et al Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2414-9; and Kohn et al Nat Med. 1998 Jul;4(7).775-80.). By way of example, a transgene may be introduced mto cells using an appropnate expression vector including but not limited to cosmids, plasmids, or modified viruses (e g replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtamed usmg standard methods including cultunng the cells on a monolayer of virus-producing cells (Van der Putten, Proc Natl Acad Sci U S A. 1985 Sep;82(18):6148-52; Stewart et al (1987) EMBO J 6:383-388). Examples of genes that may be introduced into the stem cells include genes encoding cell death protectors such as Bcl-xL and McL- 1. Cryoprotective methods can be used to maintain maximum viability of the rephcative mitochondπa. Cryopreservation can be earned out in a medium contammg for example dimethylsulphoxide, ethylene glycol, or glycerol or sucrose with 1,2-propanediol, or the mitochondna can be vitnfied using cryoprotectants such as ethylene glycol and dimethyl sulphoxide. In an embodiment of the invention, the cryopreservation procedure involves cooling the mitochondπa in a cryoprotective solution to an appropnate temperature (e.g -176°) Scanning and transmission electron microscopy can be used to assess the puπty and morphology of a preparation. In addition the preparation can be analyzed for membrane mitochondnal potential and the total number and concentration of functional mitochondπa present can be determined in accordance with conventional methods as described herem. Rephcative ability of the mitochondna in a preparation can be determined usmg conventional techniques mcludmg restnction fragment polymorphism methods as descπbed herem. The present invention generally involves the use of rephcative mitochondna to enhance the developmental potential of animal oocytes, especially mammals, mcludmg sports, zoo, pet, and farm animals, in particular dogs, cats, cattle, pigs, horses, goats, buffalo, rodents (e.g. mice, rats, guinea pigs), monkeys, sheep, and humans. In the nuclear transfer methods, rephcative mitochondπa are used to enhance the developmental potential of non-human recipient oocytes. A method of the mvention involves removmg the oocytes from follicles m the ovary. This can be accomplished by conventional methods for example, using the natural cycle, during surgical intervention such as oophorohysterectomy, during hyperstimulation protocols m an IVF program, or by necropsy. Oocyte removal and recovery can be suitably performed usmg transvaginal ultrasomcally guided folhcular aspiration. After oocytes have been isolated, rephcative mitochondna are introduced into the oocytes, or the oocytes can be cryopreserved for storage m a gamete or cell bank. If the oocytes are not cryopreserved the oocytes should be treated m accordance with the method of the mvention preferably within 48 hours after aspiration. If the oocytes are frozen, they can be thawed when it is desired to use them and treated in accordance with a method of the invention. Rephcative mitochondπa may be introduced mto the oocytes (or zygotes) by conventional micromjection techniques or by other techniques such as electrofiision of mitochondπa contained withm hposomes or other suitable means. After introduction, simultaneously with, or pnor to the introduction of the rephcative mitochondna, the oocytes are fertilized with suitable spermatozoa from the same species. The fertilization can be earned out by known techniques mcludmg sperm injection. Suitable human m vitro fertilization and embryo transfer procedures that can be used mclude m vitro fertilization (IVF) (Trounson et al. Med J Aust. 1993 Jun 21 ;158(12):853-7, Trouson and Leeton, m Edwards and Purdy, eds.. Human Conception in Vitro, New York:Academιc Press, 1982, Trounson, in Crosignam and Rubin eds., In Vitro Fertilization and Embryo Transfer, p. 315, New York: Academic Press, 1983), tntracytoplasmic sperm injection (ICSI) (Casper et al., Fertil Stenl. 1996 May;65(5):972-6); m vitro fertilization and embryo transfer (IVF-ET)(Quιgly et al, Fert. Stenl., 38: 678, 1982); gamete intrafallopian transfer (GIFT) (Molloy et al, Fertil. Stenl. 47: 289, 1987); and pronuclear stage tubal transfer (PROST) (Yovich et al., Fertil. Stenl. 45: 851, 1987). The methods and compositions of the invention can be used to mcrease the success rate of embryo development. In particular, they can be used to reduce the detrimental effects of mitochondπal DNA mutations (e.g. deletion or missense mutations) or abnormal or deficient mitochondnal function in the progeny of an individual affected by such mutations or abnormal or deficient function, by introducing in oocytes or zygotes from the mdividual rephcative mitochondπa that compnses healthy mitochondna. Mitochondπal DNA deletions or mutations usually result m impaired oxidative phosphorylation and clinical pathology related to muscle or neurologic tissues. For example Kearns- Sayre syndrome (KSS) or progressive extemal ophthalmoplegia is the result of a common 4799 bp deletion (Holt et al, Ann Neurol. 1989 Dec;26(6):699-708) and Leber's hereditary optic neuropathy (LHON) is due to a missence mutation m the mtDNA (Wallace et al, Science 242, 1427 (1998)) Therefore, mjectmg oocytes from individuals with these conditions with healthy rephcative mitochondna, creatmg heteroplasmy, may prevent the detrimental effect of mtDNA missence or deletion mutations m the progeny. The invention also contemplates unproved nuclear transfer methods usmg rephcative mitochondna. Nuclear transfer methods or nuclear transplantation methods are known in the literature and are descnbed m for example, Campbell et al, Theπogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420. Methods for isolation of recipient oocytes suitable for nuclear transfer methods are well known in the art. Generally, the recipemt oocytes are surgically removed from the ovaπes or reproductive tract of a mammal, e.g., a bovme. Once the oocytes are isolated they are πnsed and stored m a preparation medium well known to those skilled m the art, for example buffered salt solutions Recipient oocytes must generally be matured in vitro before they may be used as recipient cells for nuclear transfer. This process generally requires collectmg immature (prophase I) oocytes from mammalian ovaries, and maturing the oocytes m a maturation medium pnor to fertilization or enucleation until the oocyte attains the metaphase II stage. Metaphase II stage oocytes, which have been matured in vivo, may also be used in nuclear transfer techniques. Enucleation of the recipient oocytes may be earned out by known methods, such as descπbed in U.S Pat. No. 4,994,384 For example, metaphase II oocytes may be placed in HECM, optionally containing cytochalasin B, for immediate enucleation, or they may be placed in a suitable medium, (e.g an embryo culture medium), and then enucleated later, preferably not more than 24 hours later. Enucleation may be achieved microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm (McGrath and Solter, Science, 220:1300, 1983), or usmg functional enucleation (see U.S. 5,952,222). The recipient oocytes may be screened to identify those which have been successfully enucleated. The recipient oocytes may be activated on, or after nuclear transfer using methods known to a person skilled in the art. Suitable methods include cultuπng at sub-physiological temperatures, applymg known activation agents (e.g. penetration by sperm, electπcal and chemical shock), increasing levels of divalent cations, or reducing phosphorylation of cellular protems (see U.S. 5, 496,720) . A nucleus of a donor cell, preferably of the same species as the enucleated oocyte, is introduced into the enucleated recipient oocyte. The donor cell nucleus may be obtamed from any mammalian cells. Donor cells may be differentiated mammalian cells denved from mesoderm, endoderm, or ectoderm.. In particular, the donor cell nucleus may be obtained from epithelial cells, neural cells, epidermal cells, keraπnocytes, hematopoietic cells, melanocytes, chondrocytes, B- lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells. Suitable mammalian cells may be obtamed from any cell or organ of the body. The mammalian cells may be obtamed from different organs mcludmg skm, lung, pancreas, liver, stomach, mtestme, heart, reproductive organ, bladder, kidney and urethra. The nucleus of the donor cell is preferably membrane-bounded. A donor cell nucleus may consist of an entire blastomere or it may consist of a karyoplast. A karyoplast is an aspirated cellular subset including a nucleus and a small amount of cytoplasm bounded by a plasma membrane. (See Methods and Success of Nuclear Transplantation m Mammals, A. McLaren, Nature, Volume 109, June 21, 194 for methods for preparing karyoplasts). Rephcative mitochondna is introduced mto the enucleated recipient oocyte. The rephcative mitochondria is preferably deπved from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, and most preferably from the same mdividual from which the donor cell nucleus is deπved. Methods for preparing rephcative mitochondπa are descπbed herem. Donor cells may be propagated, genetically modified, and selected m vitro pnor to extracting the nucleus, or the rephcative mitochondna. The nucleus of a donor cell and/or the rephcative mitochondπa may be introduced mto an enucleated recipient oocyte usmg micromampulation or micro-surgical techmques known m the art (see McGrath and Solter, supra). For example, the nucleus of a donor cell may be transfeπed to the enucleated recipient oocyte by depositing an aspirated blastomere or karyoplast under the zona pellucida so that its membrane abutts the plasma membrane of the recipient oocyte. This may be accomplished using a transfer pipette. Similar methods may be used to introduce the rephcative mitochondria. Fusion of the donor nucleus and the enucleated oocyte may be accomplished according to methods known m the art. For example, fusion may be aided or induced with viral agents, chemical agents, or electro-induced. Electrofusion involves providing a pulse of electricity sufficient to cause a transient breakdown of the plasma membrane. (See U.S. 4, 994,384). In some cases (e.g. with small donor nuclei) it may be preferable to inject the nucleus directly into the oocyte rather than usmg electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264- 267 (1994). The clones produced usmg the nuclear transfer methods as descnbed herem may be cultured either in vivo (e g in sheep oviducts) or in vitro (e.g. in suitable culture medium) to the morula or blastula stage. The resulting embryos may then be transplanted mto the uteπ of a suitable animal at a suitable stage of estrus usmg methods known to those skilled m the art. A percentage of the transplants will initiate pregnancies in the stnτogate animals. The offspring will be genetically identical where the donor cells are from a single embryo or a clone of the embryo. The following non-limiting examples are illustrative of the present invention: Example 1 Injection of a mitochondnal fraction obtamed from a human myeloid cell lme (HL-60) accelerated and or facilitated preimplantation embryonic development. Muπne zygotes were microinjected with either a mitochondπal fraction or a buffer at day 0.5 and further cultured m vitro until day 3.5. Embryos receivmg mitochondπa were twice as likely to form fully expanded or hatching blastocysts when compared with the buffer injected zygotes (45% versus 17%) (See Figure 1). Example 2 Assessment of mitochondrial function, mtDNA copy number and mtDNA deletion rates in human oocytes of various ages and in human embryos showing preimplantation developmental defects. Patients with a history of either delayed embryo development (6-cell stage or less at 72 hours post insemination) or persistent embryo fragmentation resulting m only Grade 4 or 5 embryos (presence of cellular fragments filling at least 30% of total embryo volume) will be included m the study. At the time of retπeval, approximately 20% of oocytes are immature and thus unsuitable for fertilization using ICSI. These oocytes will be used in order to determine whether these patients have a maternal predisposition towards abnormal embryonic development that can be attnbuted to mitochondna. Rates of mitochondna dysfunction will be compared to immature oocytes obtamed from patients with known history of normal embryo development. In addition, fragmented embryos, unsuitable for transfer, will be analyzed and their mitochondnal status compared between these two groups. The effect of maternal age will also be determined by examining mitochondnal normality in patients aged 25-30 years, 30-35 years and 35 above. The following experiments are proposed. A/ Mitochondnal function- Changes in mitochondnal membrane potential reflect mitochondπal function since energy produced during mitochondπal respiration is stored as an electrochemical gradient across the mitochondnal membrane and is used to dπve ATP production. Disruption of mitochondπal membrane potential is one of the first signs of apoptosis m many somatic cells. Bπefly, oocytes and embryos will be incubated with a fluorochrome (DePsipher, R&D Systems) that allows simultaneous detection of mitochondπa with disrupted (non- functional) and maintained mitochondnal potential. Samples will be analyzed usmg a deconvolution microscope and the amount of fluorescence will be recorded using Delta Vision software package (Silicon Graphics). In dy g cells or those with disrupted membrane potential, the dye will remain in its monomenc form in the cytoplasm and the mitochondπa will appear green, whereas m healthy cells the dye aggregates m the mitochondna will appear red. Furthermore, this technique can be used to estimate mitochondnal copy number based on the total amount of fluorescence emitted on both channels. The immature (GV and MI stage) oocytes obtamed from the ICSI program, unfertilized oocytes from IVF, and spare embryos donated to research will be analyzed. B/ Mitochondnal copy number. In order to determine whether recurrent embryo fragmentation observed in some patients could be attnbuted to insufficient mitochondnal copy number within maternal stores, semi-quantitative PCR (Chen et al 1995 Am J Hum Genet 57, 239-47) will be used to estimate approximate mtDNA copy number. After staining and assessment of mitochondnal function, individual oocytes or embryos will be placed in 20 μl of PBS and stored m -70°C. Before PCR, samples will be boiled and 1/10 of the volume of the lysate will be used as a template for the PCR reaction C/ mtDNA deletions: Although the above studies will determine the viability and abundance of the mitochondna, a further assessment can be done using PCR to semi-quantitatively assess mtDNA deletions in the same population of human oocytes and embryos used above. Different PCR pπmer sets, encompassmg all regions of the mitochondnal chromosome, have been designed and the proportion of mitochondria with a deletion m any part of the chromosome will be determined usmg the approach of Zhang et al. (Biochem Biophys Res Commun 1996 Jun 14,223(2).450-5). This method of scanning the whole chromosome with multiple primer sets will circumvent the problems previously observed with very long mtDNA PCR (Kajander et al , Biochem Biophys Res Commun 1999 Jan 19;254(2):507-14). Preliminary results have shown that the 4799 bp common deletion can be easily identified. In addition, amplified products will be subcloned and sequenced m order to identify specific deletions that could be associated with activation of PCD. Expected Outcome. Information about mitochondπal function, mtDNA status and an estimate of mtDNA copy number will be obtained. This will allow compaπson of different oocytes and embryos m order to determme whether there might be a predisposition towards mitochondnal dysfunction m some infertile patients. This data will also be analyzed with respect to increased maternal age and confirm previous reports of a higher rate of mtDNA mutations associated with reproductive senescence Example 3 Isolation of mitochondria and mouse models of embryo demise The ability of an enriched fraction of mitochondna, isolated from both somatic cells and different types of stem cells, to enhance developmental potential and to suppress apoptosis following injection into oocytes will be assessed. The cells used for these expenments will include muπne embryonic stem (ES) cells, munne and human trophectodermal stem (TS) cells, and human or muπne CD34+/CD38- hematopoetic stem cells and granulosa cells. ES and TS cells will be grown in vitro under standard culture conditions (Hadjantonakis et al Mech Dev. 1998 Aug;76(l-2)-79-90, Tanaka et al Science. 1998 Dec 11 ,282(5396) 2072-5) The nucleated cells obtained from human umbilical cord blood of healthy donors will be isolated using a Ficoll gradient. CD34+/CD38- cells will be separated usmg a cell depletion magnetic column. Equivalent (but adult rather than fetal) cells can also be obtained from munne bone manow of adult animals (Ploemacher et al Exp Hematol 1989 Mar, 17(3).263-6) The somatic cell source will be luteimzed granulosa/cumulus cells isolated from folhcular fluid during oocyte retneval for IVF or from ovaπes of hormonally pruned mice (Trbovich et al Cell Death Differ. 1998 Jan;5(l):38-49) An ennched mitochondnal fraction can be isolated from all stem cell types and from granulosa cells usmg the method of Rickwood (Darley-Usmer VM., Rickwood D, Willson MT Mitochondna, a Practical Approach, Oxford Washmgton DC, IRL Press, 1987, pp. 1-16). Bnefly, cells are suspended m a sucrose-based buffer and lysed usmg a glass homogenizer. The nuclei are pelleted and the mitochondnal fraction is further ennched and punfied usmg a continuous Percoll gradient to separate damaged from intact mitochondπa and to eliminate most cellular debπs Scanning and transmission electron microscopy will be used to assess the puπty and morphology of the mitochondπal fraction. The maintenance of membrane mitochondπal potential will be analyzed by DePsipher dye as descπbed above m Example 1, coupled with FACS analysis for rapid calculation of the total number and concentration of both functional and damaged mitochondπa present. Only fractions containing greater than 90% functional mitochondna will be used m the subsequent studies. a) Ability of mitochondria to suppress fragmentation in FVB strain mouse oocvtes cultured in vitro Mature oocytes of FVB strain mice undergo a very high rate (~75%) of spontaneous fragmentation within 48 hours when cultured m vitro (Monta et al. Dev Biol. 1999 Sep 1;213(1):1-17). This model will be used to test each mitochondna ennched fraction for its ability to suppress oocyte fragmentation. Ovulated oocytes will be snipped of their cumulus cells and will be injected with mitochondna ennched fraction m a dose response fashion accordmg to the technique of Van Blerkom et al . (Hum Reprod. 1998 Oct;13(10):2857-68). It has been estimated that mature oocytes contain about 100,000 mitochondna (Jansen and de Boer, Mol Cell Endocnnol. 1998 Oct 25;145(l-2):81-8). Between 2000 and 20,000 mitochondna m a volume of 5 to 15 picoL will be mjected. A control group of oocytes will be left intact or mjected with either buffer used for suspension of mitochondna, or with the mitochondπa depleted fraction. Damaged mitochondπa obtamed from the percoll gradient will also be injected to determine possible negative effects of damaged mitochondπa on oocyte survival. All oocytes will then be cultured and scored for fragmentation at 24 and 48 hours. This model will be used to confirm the optimal number and type of mitochondria to inject to protect against fragmentation Expected Outcome- It is expected that mitochondπa deπved from stem cells will be successful in preventing fragmentation, and will have the benefit of potential rephcative ability. b) Does injection of mitochondria from stem cells into normal mouse zygotes fertilized in vitro provide long-lasting protection from cell death ? Increased maternal age and fertilization m vitro combmes to result in an apoptosis rate of about 30% in muπne zygotes, and to a higher cell death index at the blastocyst stage, compared to zygotes obtained from young mothers fertilized m vivo (about a 2% fragmentation rate) (Junsicova et al.. Mol Hum Reprod. 1998 Feb;4(2): 139-45). Moreover, analysis of cell death rates in human blastocysts demonstrated that approximately 30% of embryos preferentially eliminated the inner cell mass or activated cell death m the majoπty of cells. To assess if injection of mitochondna can prevent apoptosis m zygotes and also provide protection during the later developmental stages, zygotes from aged mice (ICR strain 44 weeks old) will be mjected with an ennched fraction of mitochondna and then- development to the blastocyst stage will be observed in vitro. The number of mitochondna to be injected will be estimated usmg the methods set out m the previous experiment, and the concentration will be fine tuned if necessary. At day 4.5, blastocyst cell numbers and cell death rates will be recorded, with particular attention to the inner cell mass. Further studies will examine the impact of mitochondnal injection on protection from cell death caused by vanous toxicants as an artificial digger of apoptosis. In particular, whether rmtochondnal injection can prevent apoptosis mduced by treatment with doxorubicin (Bergeron et al . 1998 Gen. Dev 12, 1304-1314), hyperglycaemia (Moley et al Nat Med 1998 Dec;4(12):1421-4) and DMBA, which have all been shown to activate the cell death pathway during blastocyst formation, will be investigated. In these experiments, zygotes injected with appropnate mitochondna will be cultured in KSOM medium until they reach the early blastocyst stage, when the experimental treatment will be performed m vitro with either doxorubicin (200nM), glucose (30mM) ennched medium or with DMBA ( lμM). Zygotes injected with buffer or with mitochondna-depleted fractions that develop to the blastocyst stage will be used as controls. At 24 hours the toxicant addition, blastocyst cell number and cell death mdex will be determined as previously descnbed (Junsicova et al . 1998, supra). Expected outcome. Somatic cell mitochondria have been shown to be diluted out by subsequent cell divisions of preimplantation embryos, and are non-detectable by the blastocyst stage (Ebert et al l 989, J Reprod Fertil Jan, 82(1) 145-9 9) Stem cell mitochondria should behave more like oocyte mitochondria, which have been demonstrated by Van Blerkom et al (Hum Reprod. 1998 Oct,13(10):2857-68) to be detectable at least 80 hours after injection mto mouse oocytes. If the donor stem-cell mitochondria are rephcative and persist to the blastocyst stage, protection from spontaneous apoptosis in vitro, and decreased rates of cell death following toxicant administration should be observed. c) Assessment of normal development of mice derived from zygotes i ected with stem-cell mitochondria To determme if mitochondna injection may compromise normal development and life span, FVB zygotes will be injected with vaπous stem or somatic cell mitochondπa-enπched fractions as described above and transfened into pseudopregnant females. At least 20 progeny in each group will be obtained The offspnng will be followed over an 18-month penod for detection of any developmental abnormalities, reproductive dysfunction, or reduced life span, that might be attributable to a deleterious effect of donor mitochondria injection on pre and postnatal development. Moreover, since 75% of oocytes from this strain normally undergo apoptosis in vitro, female offspnng will also be assessed for their oocyte fragmentation rate in vitro to determme if the donor mitochondπa have replicated in the offspring, producmg heteroplasmy. All the parameters will be compared with offspring generated from sham injected zygotes. Another way to determine the rephcative ability of donor stem-cell mitochondria is to utilize restriction fragment length polymorphism (RFLP) in mtDNA, as has been reported between strains C57B16/J and NZB/BINJ (Jackson laboratoπes) (Meirelles and Smith, Genetics 1998 Feb;148(2).877- 83). The FVB strain will be examined to determme if it contams mtDNA RFLP similar to either of the two strains and based on these results, TS or ES cell lmes will be deπved from the opposite strain. Mitochondna enriched fraction from these genetically distinct cells will be mjected mto FVB zygotes. The rephcative potential of injected mitochondna can then be confirmed m the offspnng by determining the RFLP status of the isolated mitochondna. Expected outcome. The offspring created by donor stem-cell mitochondπal injection should be phenotypically normal, with normal hfespan. These mice may have improved reproductive function, and decreased oocyte apoptosis in vitro, if the donor mitochondna are rephcative and capable of creating heteroplasmy. The ability to create heteroplasmy is cπtical to the success of any future clinical studies aimed at correcting heπtable mitochondπal diseases. D) No rescue of embryo fragmentation mediated bv DNA damage.. A subset of both male and female gametes contain damaged DNA (Sun et al , Biol Reprod. 1997 Mar;56(3).602-7, Lopes et al . Fertil Stenl 1998 Mar;69(3):528-32). Results of Twigg et al (Hum Reprod 1998 Jul;13(7).1864-71) with ROS-mduced sperm DNA damage clearly demonstrated the ability of such sperm to undergo decondensation and pronuclear formation, suggesting that early stages of embryo development may occur even if the paternal DNA is fragmented. It is not desirable to rescue embryos with chromosomal abnormalities. Genetic analysis of the cell death pathway in munne germ cells, suggests that one can prevent apoptosis m the female germ lme if the tngger is lack of survival signals, but not if the initiating factor is DNA damage. A model developed by Doerksen and Trasler (Biol Reprod 1996 Nov;55(5).l 155-62) will be used m which male mice are treated with 5-azacytidine (5-AZC), a drug that interferes with DNA methylation and induces sperm DNA damage. Female mice, when mated to these treated males, produce embryos with a high rate of fragmentation and low pregnancy rates secondary to chromosomal damage (Doerksen and Trasler, 1996, supra). In this experiment, male animals will be treated with 5-AZC (4 mg/kg for 3 weeks), sperm will be collected from the cauda epididimus and mjected together with stem cell mitochondna or buffer mto the oocytes of FVB strain mice. Expected outcome. Failure of mitochondnal injection to protect against embryo fragmentation in this model will confirm that human embryos will not be rescued m which the cell death pathway has been activated by DNA damage. In addition, the report of injection of donor oocyte cytoplasmic into the oocytes of 7 patients by Cohen and his colleagues (Lancet 1997 Jul 19,350(9072): 186-7) described 2 couples m which no improvement m embryo quality was seen These 2 couples were the only ones in which the men had severe ohgoasthenospermia, which has been shown to be associated with a high degree of sperm DNA damage (Sun et al , Biol Reprod 1997 Mar;56(3).602-7, Lopes et al , Fertil Stenl 1998 Mar;69(3).528-32, Hum Reprod 1998 Mar;13(3):703-8). The presence of DNA fragmentation in the sperm may explam why the injections were unsuccessful m these two cases. Example 4 Overexpression of Mcl-1 and Bcl-xL in stem cell mitochondria to enhance suppression of cell death in mouse and human embryos. In mouse and human oocytes and embryos, two cell death protectors, Bcl-xL and Mcl-1, both of which localize to mitochondria, are abundantly expressed. Vaπable levels of maternally stored transcnpts have been observed for these two protems in human oocytes suggesting that vanation in these protems may lead to varying susceptibility to cell death tπggers Transfected ES cells that overexpress Bcl-x L or Mcl-1, dnven by a ubiquitous chicken b-actin promoter (pCAX - Hadjantonakis et al . 1998, supra) will be created. Transfected lines will be selected based on their resistance to neomycm and will be assessed for protem levels of Mel- 1 or Bcl-x L within their mitochondπal fraction usmg western blot analysis. Cytochrome C, another mitochondπal- localized protem, will be used as a loadmg control m order to show enhanced levels of Bcl-xL and Mcl-1 m mitochondna ennched fractions. Upon establishing mcreased levels of protem expression on the mitochondπal membranes within these cells, mitochondπa will be isolated and used in similar experiments to those descnbed above. Therefore, early embryos can be augmented with more functional mitochondria, but also with mitochondna containing a higher protem content of either Bcl- x L or Mcl-1. Expected Outcome. If these transfected mitochondna are supenor m suppressing cell death compared to their non-transfected counterparts, the importance of either Bcl-x L or Mcl-1 m the prevention of apoptosis and normal embryo development m this model will be established. Example 5 Injection of mitochondria into human oocytes at the time of ICSI and rescue of fragmented embryos. Twenty patients who have undergone two cycles of IVF and who produce only very fragmented embryos (Grade 4 or 5) or embryos with delayed development (6 cells or less at 72 hours post fertilization), will be recruited for a pilot study Women must have normal day 3 serum FSH concentrations (<10 IU/L in our lab) initially, but if the results of preliminary studies appear promising, older women with elevated serum FSH concentrations will be enrolled for the procedure as well. Ovulation mduction will consist of a long GnRH-agomst protocol with vaπous human menopausal gonadotropms as previously descπbed (Greenblatt et al , Fertil Stenl. 1995 Sep;64(3):557-63). Cycles will be monitored usmg a combination of transvaginal ultrasound and serum estradiol measurements. Human chononic gonadotropin will be administered at 36 h before oocyte retπeval. Oocytes will be collected trans vaginally under ultrasound guidance. Following oocyte retneval, the cumulus cells will be removed by exposing the cumulus corona-oocyte complex to hyaluromdase m modified HTF medium. Each oocyte will be assessed for matuπty and those with a first polar body present (Mil) selected for ICSI. Immature oocytes will be used for determination of mitochondnal function and mtDNA copy number and mutations as descπbed m Example 2. Spermatozoa will be prepared on the day of oocyte retπeval as previously descπbed (Sun et al, Biol Reprod. 1997 Mar;56(3):602-7). The ICSI procedure to be used m this study has been previously descπbed m detail (Casper et al , 1996, supra). All microinjection procedures will be earned out on the heated stage of an inverted microscope (magnification x200 or x400). For the micromjections, a morphologically normal, motile sperm will be selected from a sperm/PVP droplet and immobilized. Oocytes from each patient will be divided mto two groups. Oocytes m group one will be mjected with a smgle speim as previously descnbed (Casper et al , 1996, supra). Oocytes m group 2 will be mjected with a smgle sperm aspirated mto the injection pipette together with between 5,000 and 20,000 intact mitochondna from human umbilical cord blood- derived hematopoetic stem/progenitor cells prepared as descnbed above. The volume for injection mcludmg both sperm and mitochondna will be kept to a maximum of 15 picoL. Following injection, oocytes will be transfeπed mto a 100 μl droplet of HTF medium supplemented with 5% human serum albumin m a plastic 60 x 15 mm petn dish, covered with mineral oil and mcubated m a humidified 5% CO, environment at 37°C Cultured oocytes will be assessed for the presence of two pronuclei, indicative of normal fertilization at 16-18 h after ICSI. Embryo development and gradmg according to the method of Veeck (1991; Acta Eur Fertil. 1992 Nov-Dec;23(6):275-88) will be performed daily. The embryo score (cell number X 1/grade) will be determined for each embryo at 48, and 72 hours, and cell number estimated at 96 and 120 hours. Morphologically normal appearing expanded blastocysts will be transferred at day 5 post-fertilization. If normal embryo development occurs in any of the control injected oocytes, they will be transfeπed first. The pregnancies obtamed by this technique will be followed closely and the patients advised to consider amniocentesis to rule out a gross chromosomal abnormality. Babies born as a result of this procedure will have their cord blood collected and stored for determination of mitochondnal heteroplasmy if possible (le. if a mtDNA mutation is detected in the unfertilized oocytes), and which may be responsible for the embryo fragmentation or delayed development seen initially in these patients. The babies will also be followed with assessment for normal development at birth, and at mtervals thereafter for as long as the parents agree. Expected outcome Group 1 oocytes should result m embryos with delayed development or which are completely fragmented, consistent with the patient's past history. In group 2 oocytes, injection of an ennched fraction of stem cell mitochondna will allow normal development to the blastocyst stage with lntrauteπne transfer and pregnancy in some patients. The present invention is not to be limited in scope by the specific embodiments descnbed herem, smce such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, vanous modifications of the invention m addition to those shown and descπbed herein will become apparent to those skilled in the art from the foregomg descnption. Such modifications are mtended to fall within the scope of the appended claims. All publications, patents and patent applications refeπed to herem are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually mdicated to be incorporated by reference m its entirety. All publications, patents and patent applications mentioned herem are incorporated herem by reference for the purpose of descnbmg and disclosing the methodologies etc. which are reported therein which might be used in connection with the mvention. Nothing herem is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of pnor invention. It must be noted that as used herem and m the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" includes a plurality of such genes.

Claims: WE CLAIM: 1 A method for enhancing developmental potential of oocytes or zygotes compπsing increasing intracellular levels of rephcative mitochondna m oocytes or zygotes. 2. A method as claimed in claim 1 wherein the intracellular levels of rephcative mitochondπa are increased by mtroducmg rephcative mitochondna denved from stem cells or an immortalized cell line. 3. A method as claimed in claim 2 wherem the rephcative mitochondπa are introduced by microinjection or electrofusion. 4. A method as claimed in claim 2 or 3 wherein the stem cells have been genetically modified. 5. A method as claimed in claim 1, 2 or 3 wherem the rephcative mitochondria compnse mitochondπal DNA free of deletions or mutations. 6. A method as claimed in any of the precedmg claims wherein the rephcative mitochondria are at least 60% free, preferably 75% free, and most preferably 90% free from other cytoplasmic components. 7 A method as claimed m claim 2 wherein the rephcative mitochondna deπved from the stem cells or immortalized cell line contains about 2,000 to 20,000 mitochondπa. 8. A method as claimed m any of the precedmg claims wherein the ooctyes or zygotes are from sports, zoo, pet and farm animals. 9. A method as claimed m any of the precedmg clauns wherem the developmental potential of human ooctyes are enhanced 10. An oocyte or zygote with increased intracellular levels of mitochondna obtamed from a method as claimed in any of the preceding claims. 1 1 A method as claimed m claim 9 further compnsmg fertilizing the oocytes to obtam a zygote with increased intracellular levels of rephcative mitochondna. 12. A zygote with increased intracellular levels of mitochondπa obtamed from a method as claimed m claim 11. 13. A composition compnsmg rephcative mitochondπa for enhancmg developmental potential of oocytes and zygotes. 14. A composition as claimed m claim 13 wherem the rephcative mitochondna is deπved from stem cells or an immortalized cell line. 15. A composition as claimed in claim 13 wherem the rephcative mitochondna is denved from differentiated mammalian cells. 16. A method for reducmg the detrimental effects of rmtochondnal DNA mutations m the progeny of an individual affected by such mutations compnsmg mtroducmg mto oocytes or zygotes from the individual rephcative mitochondna compnsmg healthy mitochondna. 17. A method as claimed in claim 16 wherein the rephcative mitochondna compnse mitochondrial DNA free of deletions or mutations resulting m impaired oxidative phosphorylation and clinical pathology related to muscle or neurologic tissues. 18. A method for improvmg embryo development after m vitro fertilization or embryo transfer m a female mammal compπsmg implanting mto the female mammal an embryo deπved from an ooctye or zygote contammg increased intracellular levels of rephcative mitochondria. 19 A method of cloning a non-human mammal by nuclear transfer compnsmg (a) introducing a donor cell nucleus denved from donor cell of a non-human mammal, and rephcative mitochondna preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same non- human mammal from which the donor cell nucleus is denved, mto an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) cultuπng the nuclear transfer unit to provide an embryo, (c) implanting the embryo into the uterus of a sunogate mother of said species, and (d) permitting the embryo to develop mto the cloned mammal. 20 A method to produce viable embryos of a non-human mammal compπsing: (a) introducing a donor cell nucleus denved from a donor cell of a non-human mammal, and rephcative preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same non-human mammal from which the donor cell nucleus is deπved, mto an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) cultunng the nuclear transfer unit to provide an embryo 21 A method of clonmg a fetus of a non-human mammal by nuclear transfer compπsmg the followmg steps- (a) introducing a donor cell nucleus denved from a donor cell of a non-human mammal, and rephcative preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same non-human mammal from which the donor cell nucleus is denved, mto an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit, (b) cultuπng the nuclear transfer unit until greater than the 2-cell developmental stage; and (c) transferring the cultured nuclear transfer unit to a host non-human mammal of the same species such that the nuclear transfer unit develops mto a fetus. 22. A method as claimed m claim 21, wherein the fetus develops mto an offspnng 23. A method as claimed m any one of claims 19 to 22, wherem the donor cell nucleus is from mesoderm, endoderm, or ectoderm. 24 A method as claimed m any one of clauns 19 to 23 wherem the non-human mammal is bovme, ovme, porcine, equme, caprine and buffalo. 25. A method as claimed in any one of clauns 19 to 24, wherem the donor cell nucleus is from epithehal cells, neural cells, epidermal cells,kerahnocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, or muscle cells. 26. A method as claimed m any one of clauns 19 to 25, wherem the donor cell nucleus is from an organ selected from the group consistmg of skm, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra. 27. A method as claimed m any one of clauns 19 to 26, wherem the enucleated recipient oocyte is matured in vitro or in vivo pπor to enucleation. 28. A method as claimed m any one of clauns 19 to 27 wherem the enucleated recipient oocyte is a Metaphase II stage oocyte. 29. A method as claimed in any one of claims 19 to 28 wherein the donor nucleus is membrane- bounded 30. A method as claimed in any one of clauns 19 to 29 wherein the donor nucleus is a whole blastomere 31. A method as claimed in any one of clauns 19 to 30 wherem the donor nucleus is a karyoplast aspirated from a blastomere. 32. A recipient oocyte compπsmg a penvitelline space, and a donor cell nucleus and rephcative mitochondπa deposited m the penvitelline space. 33. A recipient oocyte as claimed in claim 32 wherem the rephcative mitochondπa is denved from the same species and cell type as the donor cell nucleus or from the same mdividual from which the donor cell nucleus is denved.

Inventors: CASPER ROBERT F, JURISICOVA ANDREA, PEREZ GLORIA I, ROGERS IAN, TILLY JONATHAN, CASPER, ROBERT, F., JURISICOVA, ANDREA, PEREZ, GLORIA, I., ROGERS, IAN, TILLY, JONATHAN
Applicants: CASPER ROBERT F, GEN HOSPITAL CORP DOING BUSINE, JURISICOVA ANDREA, MOUNT SINAI HOSPITAL CORP, PEREZ GLORIA I, ROGERS IAN, TILLY JONATHAN, CASPER, ROBERT, F., JURISICOVA, ANDREA, MOUNT SINAI HOSPITAL, PEREZ, GLORIA, I., ROGERS, IAN, THE GENERAL HOSPITAL CORPORATION DOING BUSINESS AS MASSACHUSETTS GENERAL HOSPITAL, TILLY, JONATHAN
Application year: 2000
Main classification: C12N5
IPC codes: A01K67, A61K35, C12N5, C12N15