JP2003512833A - METHODS AND COMPOSITIONS FOR ENHANCED DEVELOPMENTAL CAPABILITIES OF OOCYTES AND ZYGOTS - Google Patents

Abstract

  (57) [Summary] The present invention relates to compositions and methods for enhancing the developmental potential of an egg cell or zygote by increasing the intracellular amount of replicating mitochondria in the egg cell or zygote. In one aspect of the invention, the intracellular amount of replicating mitochondria is increased by introducing replicating mitochondria into the egg cell or zygote. Oocytes can be fertilized to obtain zygotes with an increased intracellular amount of replicating mitochondria. The methods and compositions can be used to improve in vitro fertilization and embryo transfer and nuclear transfer methods.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION

The present invention is directed to egg cells, zygotes and preimplantation.
compositions and methods for enhancing the developmental capacity of embryos).

[0002]

BACKGROUND OF THE INVENTION

Using in vitro fertilization (IVF) and other assisted reproduction methods, approximately 50% of human embryos undergo a suicide program of active cell death and become fragmented. Some infertile patients produce only fragmented embryos, which is likely to be the cause of their inability to become pregnant or to maintain their pregnancy to term.

In all mammals, including humans, zygote development and primary cleavage are maternal RNA
And, depending on the protein product deposited during oogenesis. Reproductive failure can be attributed to lack of division in the developing embryo. This phenomenon can be traced to defects in the cytoplasmic composition of egg cells. The maternal cytoplasmic component is involved in embryonic arrest, which is transferred to the "2-cell block" in mice by transplanting the cytoplasm from the non-arrested strain zygotes into the zygotes of the arrested strain. ) ”Can be overcome (Muggleton-Harris et a
l. Nature, 1982 Sep 30; 299 (5882): 460-2.
). Similar egg cell cytoplasmic transfer experiments showed an improvement in the viability of immature eggs in mice (Flood et al., 1990). Later, Cohe
n and colleagues obtained pregnancy by transferring the cytoplasm of donor egg cells into the patient's egg cells at the time of intracytoplasmic sperm injection in patients with a consistently fragmented embryo history (Cohen et al. , Lancet, 1997 Jul
19; 350 (9072): 186-7). More recently, Lanzendorf
et al. (Fertil Steril. 1999 Mar; 71 (3).
: 575-7) microinjected into egg cells, where the cytoplasm of freeze-thawed egg cells enhances the developmental capacity of the egg cell after fertilization and previously created twin pregnancy in patients who had only fragmented embryos. It has occurred.

Nuclear transfer as a means of producing the same solid (clone) has been successfully carried out in several mammalian species including goat, sheep, pig, cow and mouse. In all of these cases, the efficiency of this method is very low. The reconstructed embryo develops moderately to the blastocyst stage (-40%, Ogura et al, 2000 Biol.
Reprod. 62 (6): 1579-84, 2000; Mol. Reprod
． Development 57: 55-59, 2000), the survival birth rate is unexpectedly low (1 to 7%, Ogaua et al, 2000, ibid, Pole.
jaeva et al Nature 2000 Sep 7; 407 (68).
00): 86-90). Moreover, extensive fetal and early neonatal mortality was previously reported in pups obtained from nuclear transfer (Rideout WM 3r.
d, Wakayama T, Wutz A, Eggan K et al 20.
00 Nat Geneb Feb; 24 (2): 109-10).

Thus, there is a need to enhance the developmental capacity of egg cells and improve reproductive methods, including nuclear transfer methods.

[0006]

[Outline]

The present invention relates to a method for enhancing the developmental capacity of an egg cell, which comprises increasing the intracellular amount of replicating mitochondria in the egg cell. In one embodiment of the invention, introducing replicating mitochondria into egg cells increases the intracellular mass of replicating mitochondria. The method of the present invention may further comprise fertilizing an egg cell to obtain a zygote having an increased intracellular amount of replicating mitochondria.

The present invention also relates to a method for enhancing the developmental capacity of a zygote comprising increasing the intracellular mass of replicating mitochondria in the zygote. In one aspect of the invention, introducing replicating mitochondria into the zygote increases the intracellular mass of replicating mitochondria.

The invention further relates to egg cells or zygotes with increased intracellular amount of replicating mitochondria obtained from the method of the invention.

In yet another aspect, the invention relates to a composition comprising replicating mitochondria for enhancing the developmental capacity of egg cells or zygotes, and for the treatment and prevention of inherited mitochondrial disorders. The composition may include cryopreserved mitochondria.

[0010] In another aspect, the invention provides a method for fertilizing an egg cell, comprising removing the egg cell from an ovarian follicle, introducing replicating mitochondria into the egg cell, and fertilizing the resulting egg cell with sperm. I will provide a.

In yet another aspect, the present invention comprises cryopreserving an immature egg cell, thawing the cryopreserved egg cell, and introducing replicating mitochondria into the egg cell, and then preserving the egg cell for development. Provide a way to build capacity. Also included are methods for enhancing the developmental capacity of egg cells, including cryopreserving replicating mitochondria, thawing mitochondria, and introducing replicating mitochondria into egg cells.

The methods and compositions of the present invention improve the quality of fertilized egg cells and the quality of zygotes, and improve the success rate of embryo development and ongoing pregnancy. The methods and compositions are particularly useful in enhancing the developmental capacity of egg cells or zygotes with mitochondrial DNA mutations or aberrant mitochondrial metabolic activity.

In one aspect, the invention comprises implanting an embryo derived from an egg cell or zygote containing increased intracellular mass of replicating mitochondria into a female mammal,
Provided are methods for improving embryonic development following in vitro fertilization or embryo transfer in female mammals.

The present invention relates to an individual affected by a mitochondrial DNA mutation, which comprises introducing replicating mitochondria containing no DNA mutation (ie, healthy mitochondria) into an egg cell or zygote from the individual. Also provided are methods for reducing the deleterious effects of mitochondrial DNA mutations (eg, deletions or missense mutations) in the offspring of. The present invention further provides an egg cell or zygote that contains both mitochondria having a mitochondrial DNA mutation as well as purified and isolated replicative mitochondria that do not contain a mitochondrial DNA mutation (ie, healthy mitochondria).

The present invention provides for the effect of inherited mitochondrial disorders involving the introduction of replicating mitochondria, including mitochondria that do not contain DNA mutations (ie, healthy mitochondria), into egg cells or zygotes from an individual. It also relates to methods for treating an inherited mitochondrial disease in an individual who has received the disease.

In an aspect of the invention, the egg cell is the recipient egg cell in the nuclear transfer method. The present invention thus relates to a method for enhancing the developing capacity of a recipient egg cell in a nuclear transfer method, which comprises introducing replicating mitochondria into the recipient egg cell. The invention also includes recipient egg cells containing replicating mitochondria as well as undifferentiated embryo cells, embryos and non-human animals formed from the nuclear transfer method of the invention. In the conventional nuclear transfer method, donor nuclei are placed in enucleated egg cells obtained from various solids. Thus, mitochondria in recipient egg cells have never coexisted with donor nuclei. Since mitochondria are always maternally inherited, their replication, transcription, translation and function are not only dependent on mitochondrial DNA, but the nuclear genome coexisting with mitochondria is tightly intercalated. The present invention enhances the developing ability of a recipient egg cell by introducing replicating mitochondria into the recipient egg cell. This is expected to improve the survival birth rate in the nuclear transfer method.

In one embodiment, the invention comprises (a) a donor cell nucleus derived from a non-human mammalian donor cell and preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, Preferably, replicating mitochondria from a non-human mammal from which the donor cell nucleus is derived is introduced into an enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) the nuclear transfer unit is cultured. Providing a method of cloning a non-human mammalian embryo by nuclear transfer, which comprises forming an embryo.

The method may further include allowing the embryo to develop into a cloned mammal. Accordingly, the present invention comprises (a) a donor cell nucleus derived from a non-human mammalian donor cell and 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 donor cell nucleus. Introducing replicating mitochondria from a non-human mammal into a non-human mammal enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) culturing the nuclear transfer unit to embryo (C) cloning a non-human mammal by nuclear transfer, including implanting the embryo in the uterus of a surrogate mother of the species and (d) allowing the embryo to develop into the cloned mammal. It also provides a method.

In yet another embodiment, (a) a donor cell nucleus derived from a non-human mammalian donor cell and preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, Most preferably, replicating mitochondria from a non-human mammal from which the donor cell nucleus is derived are introduced into an enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) 2 nuclear transfer units -Culture until larger than the cell development stage; (c) non-nuclear translocation, including the step of transposing the cultured nuclear translocation unit to a host non-human mammal of the same species to develop the nuclear translocation unit into the fetus. A method for cloning a human mammalian fetus is provided.

[0020]   The method can also include developing the fetus into the offspring.

In yet another aspect, the invention is most preferably from the perivitelline space and donor cell nuclei deposited in the perivitelline space and preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell. Provide a recipient egg cell containing replicative mitochondria from the same individual from which the donor cell nucleus was derived.

Other objects, features and advantages of the invention will be apparent from the detailed description below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, which is the spirit of the invention. And various changes and modifications within the scope will be apparent to those skilled in the art.

[0023]

Detailed Description of the Invention

The term "egg cell" refers to gametes from the follicles of female animals, whether vertebrate or invertebrate. Preferably the animal is a mammal, more preferably a non-human primate, cow, horse, pig, sheep, goat, buffalo, guinea pig, hamster, rabbit, mouse, rat, dog, cat or human. Egg cells suitable for use in the present invention include immature egg cells, and in vitro or in vivo with egg cell donors, mating factors (fertility agents) or fertility enhancing factors (eg, inhibin, inhibin and activin, clomiphene citrate, Included are mature oocytes from the ovary stimulated by administration of human menopausal urinary gonadotropins containing FSH or a mixture of FSH and LH and / or human chorionic gonadotropins. In some aspects of the invention, the egg cells are senescent (eg, from humans older than 40 years or from animals past their heyday of reproduction). The egg cell in some embodiments of the invention contains a mitochondrial DNA mutation. Methods for isolating egg cells are known in the art.

In the nuclear transfer aspect of the invention, egg cells are used as recipient cells (such cells are referred to herein as "recipient egg cells"). Recipient egg cells include non-human mammals, especially livestock, sport animals, zoo animals including but not limited to cows, sheep, pigs, horses, goats, buffalo and guinea pigs, rabbits, mice, hamsters, rats, vertebrates and the like. And obtained from pet animals.

[0025]   The term "zygote" refers to a fertilized egg cell before the first cleavage.

The expression “enhanced developmental capacity of an egg cell” refers to improving the quality of the egg cell so that it becomes more fertile, and / or the function or activity of mitochondria in the egg cell for subsequent development and reproduction. It means strengthening. The improved egg cell quality and thus the fertilized egg cell (eg, zygote) preferably results in the development of the egg cell into the embryo and its enhanced ability to be implanted to form healthy offspring. The phrase "enhancing the developmental capacity of the zygote" refers to improving the quality of the zygote and / or enhancing the function or activity of mitochondria in the zygote for subsequent development and reproduction. The improved quality of the zygotes preferably results in the development of the zygotes into the embryo as well as their enhanced ability to be implanted to form healthy offspring. Quality can be assessed by visual appearance of developing embryos and by IVF or nuclear translocation success rate. Criteria for determining the quality of developing embryos by visual means include, for example, their shape, rate of cell division, fragmentation, cytoplasmic appearance and other means recognized in the art of IVF and nuclear transfer. included.

[0027]   "Sperm" refers to a male gamete that can be used to fertilize an egg cell.

“Inherited mitochondrial disease” refers to a disease caused by defects in mitochondrial DNA or by defects in nuclear genes important for mitochondrial function. Examples of mitochondrial diseases are Keynes-Sayre syndrome, MERRF syndrome (myoclonus epilepsy with defective red muscle fibers), M
ELAS syndromes, including but not limited to mitochondrial encephalopathy, myopathy, lactic acidosis and stroke-like episodes, and Leber's disease (I).
． Nonaka, Current Opinion in Neurology
and Neurosurgery, 5 (1992) 622).

The term “replicating mitochondria” refers to a purified mitochondrial preparation capable of replicating and increasing mitochondrial copy number or function during embryonic development. Replicating mitochondria are substantially free of nuclear DNA, mRNA, proteins, antioxidants and other cytoplasmic components including organelles other than mitochondria.
The replicating mitochondrial preparation is free of at least 60% other cytoplasmic components, preferably 75%, and most preferably 90%. Preferably, the replicating mitochondrial preparation contains more than 70%, more preferably more than 80% and most preferably more than 90% functional mitochondria. Replicative mitochondrial preparations typically have 5-15 mitochondria of approximately 2,000-20,000.
It is contained in the volume of Pico L.

For the non-nuclear-metastatic aspect of the invention, the replicating mitochondria are preferably immortalized in any species, preferably human stem cells (eg hematopoietic, embryonic, villous, primordial germ cells). Derived from a cell line such as cancer or a deliberately transformed somatic cell. The cells are preferably a common mitochondrial deletion mutation clinically found in patients with KSS syndrome (ie 4 in nt 8470-13,477).
No deletion of the 799 bp region; see Simonnetti et al, 1992) or other pathological mitochondrial DNA mutations. Specific methods for the preparation of replicating mitochondria are exemplified herein and in Darley-Usmer VM. , Rickwood D, Willson
MT. Mitochondria, a Practical Approach
, Oxford Washington DC. , IRL Press, 198
7, pp. 1-16.

The stem cells used to prepare replicative mitochondria can be genetically modified by genetic engineering methods. Calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection,
The transgene can be introduced into cells via conventional methods such as electroporation or microinjection. Suitable methods for transforming and transfecting cells are described in Sambrook et al. (Molecu
Lar Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laborator
y press (1989)) as well as other laboratory textbooks. (Nolta et al Blood. 1995 Jul 1; 86 (1).
: 101-10; and Nolta et al Proc Natl Acad.
Sci USA. 1996 Mar 19; 93 (6): 2414-9;
And Kohn et al Nat Med. 1998 Jul; 4 (7): 7.
See also 75-80). Suitable expression vectors, including but not limited to cosmids, plasmids or modified viruses (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) can be used to introduce the transgene into cells. Transfection is easily and effectively obtained using standard methods involving culturing cells on a monolayer of virus-producing cells (
Van der Putten, Proc Natl Acad Sci U
S A. 1985 Sept; 82 (18): 6148-52; Stewart.
et al. (1987) EMBO J. 6: 383-388). Examples of genes that can be introduced into stem cells include genes encoding cell death protectors such as Bcl-xL and McL-1.

Cryoprotection can be used to preserve maximal viability of replicating mitochondria. For example, cryopreservation can be performed in a medium containing glycerol or sucrose with dimethylsulfoxide, ethylene glycol or 1,2-propanediol, or mitochondria can be treated with a cryoprotectant such as ethylene glycol and dimethylsulfoxide. Can be vitrified. In an embodiment of the invention, the cryopreservation method comprises cooling mitochondria in a cryoprotective solution to a suitable temperature (eg -176 ^° C).

Scanning and transmission electron microscopy analyzes can be used to assess the purity and morphology of the preparations. In addition, according to the usual methods described herein, the membrane mitochondrial potential.
The preparation can be analyzed for al) and the total number and concentration of functional mitochondria present can be determined. The mitochondrial replicative capacity in a preparation can be determined using conventional methods, including the restriction fragment polymorphism method described herein.

The present invention generally relates to animal egg cells, in particular mammals, including athletics, zoos, pets and farm animals, in particular dogs, cats, cows, pigs, horses, goats, buffaloes, mice (eg mice, Rat, guinea pig), monkey, sheep, and human including the use of replicating mitochondria to enhance the developmental capacity of egg cells. In the nuclear transfer method,
Replicating mitochondria are used to enhance the developmental capacity of non-human recipient oocytes.

The method of the present invention involves removing egg cells from follicles in the ovary. The hyperstimulation protocol in the IVF program during routine surgical interventions such as ovariectomy is performed by conventional methods.
This can be done using a natural cycle during ols) or by autopsy. Transvaginal ultrasound-induced follicular aspiration can be used to properly remove and recover egg cells.

After the egg cells are isolated, replicating mitochondria can be introduced into the egg cells, or the egg cells can be cryopreserved for storage in gametes or cell banks. If the egg cells are not cryopreserved, they should be processed according to the method of the invention, preferably within 48 hours after aspiration. If the egg cells are frozen, they can be thawed when desired and used according to the method of the invention.

Introducing replicating mitochondria into egg cells (or zygotes) by conventional microinjection methods or by alternative methods such as electrofusion of mitochondria contained within liposomes or other suitable means. You can

After the introduction of replicating mitochondria, at the same time as or before, the egg cells are fertilized with suitable sperm from the same species. Fertilization can be performed by known methods, including sperm injection. Suitable human in vitro fertilization and embryo transfer methods that can be used include in vitro fertilization (IVF) (Trouson et al. Med J Aust. 199).
3 Jun 21; 158 (12): 853-7, Trouson and L.
eeton, in Edwards and Purdy, eds. , Huma
n Concept in Vitro, New York: Acade
mic Press, 1982, Trounson, in Crosignan
i and Rubin eds. , In Vitro Fertilizer
ion and Embryo Transfer, p. 315, New Yo
rk: Academic Press, 1983); Intracytoplasmic sperm injection (ICS)
I) (Casper et al., Fertil Steril, 1996.
May; 65 (5): 972-6); in vitro fertilization and embryo transfer (IVF-ET).
(Quigly et al, Fert. Steril., 38: 678, 19).
82); Gamete intra-Fallopian transition (GIFT) (Molloy et al,
Fertil. Steril. 47: 289, 1987); and pronucleus stage fallopian tube metastasis (PROST) (Yovich et al., Fertil. Steril).
． 45: 851, 1987).

The methods and compositions of the present invention can be used to improve the success rate of embryo development.
In particular, by introducing them into replicating mitochondria, including healthy mitochondria, in egg cells or zygotes from individuals, in solid offspring affected by mitochondrial DNA mutations or aberrant or insufficient function, It can be used to reduce the adverse effects of mitochondrial DNA mutations (eg deletions or missense mutations) or abnormal or insufficient mitochondrial function.

Mitochondrial DNA deletions or mutations usually result in impaired oxidative phosphorylation and clinical pathology associated with muscle or nervous tissue. For example, Keens-Sayre syndrome (KSS) or progressive external ophthalmoplegia is the result of a common 4799 bp deletion (Holt et al., Ann Neurol. 1989 Dec;
26 (6): 699-708), Leber's inherited optic neuropathy (LHON) is m.
Due to a missense mutation in tDNA (Wallace et al.,
Science 242, 1427 (1998)). Therefore, injecting healthy replicating mitochondria into egg cells from individuals with these conditions to create heteroplasmy can prevent the adverse effects of mtDNA missense or deletion mutations in progeny.

The present invention also includes an improved method of nuclear transfer using replicating mitochondria. Nuclear transfer or nuclear transfer methods are known in the literature, eg Campbell et
al, Theriogenology, 43: 181 (1995); Colla.
s et al, Mol. Report Dev. , 38: 264-267 (1
994); Keefer et al, Biol. Reprod. , 50:93
5-939 (1994); Sims et al, Proc. Natl. Aca
d. Sci. , USA, 90: 6143-6147 (1993); WO 94 /.
26884; WO 94/24274, WO 90/03432, US Patent No. 4
, 944, 384 and 5,057, 420.

Methods for isolating recipient egg cells suitable for nuclear transfer methods are well known in the art. Generally, the recipient egg cell is surgically removed from the ovary or reproductive tract of a mammal, such as a bovine. Once the egg cells are isolated, they are rinsed and stored in a preparation medium well known to those skilled in the art, such as a buffered salt solution.

Recipient egg cells generally must be matured in vitro before they can be used as recipient cells for nuclear transfer. This method generally involves collecting immature (early I) egg cells from a mammalian ovary and allowing the egg cells to mature into a maturation medium prior to fertilization or enucleation.
(m medium), requires that the egg cell matures until it reaches metaphase II stage. In vivo matured stage II egg cells can also be used in the nuclear transfer method.

Enucleation of the recipient egg cells can be performed by known methods such as those described in US Pat. No. 4,994,384. For example, for immediate enucleation of metaphase II egg cells, they can be placed in HECM, which can optionally contain cytochalasin B, or they can be placed in a suitable medium (eg, embryo culture medium). It can be placed and then enucleated later, preferably within 24 hours. Microsurgery using a micropipette to remove polar bodies and adjacent cytoplasm (McGr
ath and Solter, Science, 220: 1300, 1983.
), Or functional enucleation (see US Pat. No. 5,952,222). Recipient egg cells can be screened to identify those that have been successfully enucleated.

Recipient egg cells can be activated at the time of nuclear transfer, or thereafter, using methods known to those of skill in the art. Suitable methods include culturing below physiological temperature, application of known activators (eg osmosis by sperm, electrical and chemical shock), increasing the amount of divalent cations, or phosphorylation of cellular proteins. (See US Pat. No. 5,496,720).

Preferably, the nucleus of a donor cell of the same species as the enucleated egg cell is introduced into the enucleated recipient egg cell. Donor cell nuclei can be obtained from any mammalian cell. Donor cells can be differentiated mammalian cells derived from mesoderm, endoderm or ectoderm. Specifically, donor cell nuclei are derived from epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts and muscle cells. Obtainable. Suitable mammalian cells can be obtained from any cell or body organ. Mammalian cells can be obtained from various organs including skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney and urethra.

The nucleus of the donor cell is preferably membrane-bound. The donor cell nucleus can consist of whole blastomeres, or it can consist of nucleoplasm. The nucleoplasm is a subset of aspirated cells containing a small amount of cytoplasm bound by the nucleus and the plasma membrane (
subset). (Regarding preparation method of nucleoplasm, Method and Suc
cess of Nuclear Transplantation in M
ammals, A .; McLaren, Nature, Volume 109, J
une 21, 194).

Replicative mitochondria are introduced into enucleated recipient oocytes. The replicating mitochondria are preferably derived 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 individual from which the donor cell nucleus is derived. Methods for preparing replicating mitochondria are described herein.

Donor cells are grown in vitro prior to extraction of nuclear or replicating mitochondria,
It can be genetically modified and selected.

Nuclear and / or replicating mitochondria of donor cells can be introduced into enucleated recipient oocytes using micromanipulation or microsurgical methods known in the art (see McGrath and Solter, supra). I want to be). For example, the nucleus of the donor cell can be transferred to the enucleated recipient egg cell by depositing aspirated blastomere or nucleoplasm under the zona pellucida and contacting the membrane with the plasma membrane of the recipient egg cell. This can be done using a transfer pipette. Similar methods can be used to introduce replicating mitochondria.

Fusion of the donor nucleus and the enucleated egg cell can be performed according to methods known in the art. For example, the fusion can be assisted or induced with viral agents, chemical agents, or electro-induced. Electrofusion involves applying a pulse of electricity sufficient to cause a transient disruption of the plasma membrane. (U
． S. 4,994,384). In some cases (eg with small donor nuclei) it may be preferable to inject the nuclei directly into the egg cell without electroporation fusion. Such a method is known as Collas an
d Barnes, Mol. Reprod. Dev. , 38: 264-267 (
1994).

Clones made using the nuclear transfer method described herein are cultured in vivo (eg, in oviduct sheep) or in vitro (eg, in a suitable medium) to the morula or blastula stage. be able to. The resulting embryos can then be transplanted into the uterus of a suitable animal at a suitable stage of estrus using methods known to those of skill in the art. A percentage of the implant will initiate pregnancy in the surrogate. If the donor cells are from one embryo or clone of an embryo, the offspring will be genetically identical.

[0053]   The following non-limiting examples are illustrative of the present invention:

[0054]

【Example】

Example 1 Injection of mitochondrial fraction obtained from a human bone marrow cell line (HL-60) accelerated and / or facilitated preimplantation embryo development. Mouse zygotes were injected under microscope with either mitochondrial fraction or buffer on day 0.5.
It was further cultured in vitro for up to 5 days. Embryos receiving mitochondria were twice as likely to form fully swelling or hatching blastocysts when compared to buffer-injected zygotes (45% vs. 17%) (see Figure 1). I want to be). In human oocytes and human embryos of different ages showing a growth defect in Example 2 preimplantation, mitochondrial function, evaluated late embryonic development of mtDNA copy number and mtDNA Ketsushitsuritsu (72 hours after fertilization 6- Sub-cell stage) or grade 4 or 5 only embryos (presence of cell fragments that fill at least 30% of the total embryo volume)
Patients with a history of continued embryo fragmentation resulting in are included in the study. At the time of harvest, approximately 20% of the oocytes are immature and thus unsuitable for fertilization using ICSI. These oocytes are used to determine if these patients have a maternal predisposition to abnormal embryonic development that can be attributed to mitochondria. Mitochondrial dysfunction rates are compared to immature oocytes obtained from patients with a history of normal embryo development. In addition, fragmented embryos unsuitable for transplantation will be analyzed to compare their mitochondrial status between these two groups. The effects of maternal age are also determined by examining mitochondrial normality in patients aged 25-30, 30-35 and above 35 years. The following experiments are proposed. A / mitochondrial function Changes in mitochondrial membrane potential are a function of mitochondria because the energy generated during mitochondrial respiration is stored as an electrochemical gradient across the mitochondrial membrane and used to drive ATP production. To reflect. Mitochondrial membrane potential arrest is one of the first signs of somatic cell apoptosis. Briefly, oocytes and embryos have fluorescent dyes (that enable simultaneous detection of mitochondria by arrested (non-functional) and maintained mitochondrial potential (
DePsipher, R & D Systems). Decondensing the sample (deconvoluti
on) Analyze using a microscope and record the amount of fluorescence using the Delta Vision software package (Silicon Graphics). In dying cells or cells with an arrested membrane potential, the dye will stop in its monomeric form in the cytoplasm and the mitochondria will appear green, whereas in healthy cells the pigment that aggregates in the mitochondria will It will look red. In addition, this method can be used to calculate mitochondrial copy number based on the total amount of fluorescence emitted on both channels. Immature (GV and MI stage) oocytes obtained from the ICSI program, unfertilized oocytes from IVF and spare embryos donated for study are analyzed. B / Mitochondrial copy number To determine whether recurrent embryo fragmentation observed in some patients could be attributed to insufficient mitochondrial copy number in maternal depots, a semi-quantitative PCR ( Chen et al. 1995 Am J Hum Genet 57, 239-47) is used to calculate approximate mtDNA copy numbers. After staining and evaluation of mitochondrial function, individual oocytes or embryos are placed in 20 μl PBS and stored at -70 ° C. Prior to PCR, boil the sample and PCR 1/10 of the volume of lysate
Used as a template for the reaction. C / mtDNA Deletion Although the above studies will determine mitochondrial viability and abundance,
Further evaluation can be performed using PCR to semi-quantitatively evaluate mtDNA deletions in the same population of human oocytes and embryos used above. Select a set of different PCR primers that cover all regions of the mitochondrial chromosome, and determine the percentage of mitochondria with deletions in any part of the chromosome.
g et al. (Biochem Biophys Res Commun 1996 Jun 14; 223 (2): 450-5). This method of scanning the entire chromosome, including multiple sets of primers, is based on PCR of very long mtDNA (Kajander et al;., Biochem Bioph
ys Res Commun 1999 Jan 19; 254 (2): 507-14) will avoid the problems previously acknowledged. Preliminary results showed that a common deletion of 4799 bp could be easily identified. In addition, the amplified products are subcloned and sequenced to identify specific deletions that can be associated with activation of PCD.

The expected results will provide information on mitochondrial function, mtDNA status and mtDNA copy number calculations. This will allow comparison of different oocytes and embryos to determine if they are predisposed to mitochondrial dysfunction in some infertile patients. This data was also analyzed for the older age of the mother,
It will corroborate previous reports of higher rates of mtDNA mutations associated with aging pregnancy. Example 3 Isolation of mitochondria and mouse model of embryonic death To enhance developmental potential and suppress apoptosis after injection into oocytes,
The capacity of the concentrated fraction of mitochondria isolated from both somatic cells and different types of stem cells is calculated. The cells used in these experiments were mouse embryonic stem (ES) cells, mouse and human trophectoderm stem (TS) cells and human or mouse CD.
Includes 34 + / CD38- hematopoietic stem cells and granulosa cells. ES and TS cells will grow in vitro under standard culture conditions (Hadjantonakis et al, Mech Dev.
1998 Aug; 76 (1-2): 79-90, Tanaka et al. Science, 1998 Dec 11; 282 (5396): 2072-
Five). Nucleated cells from healthy donor human cord blood are isolated using a Ficoll gradient. CD34 + / CD38- cells are separated using a cell-depleted magnetic column. Equivalent (but not fetal, mature) cells can also be obtained from adult animal mouse bone marrow (Ploemacher et al. Exp Hematol, 1989 Mar; 17 (3): 263-6.
). Somatic cell sources may be luteinized granulosa / cumulus cells isolated from the ovary of mice primed with follicular fluid or hormones during IVF oocyte collection (Trbovich et al. Cell Death Differ. 1998 Jan; 5 (1): 38-49). The enriched mitochondrial fraction was derived from whole stem cell type and granulosa cells by the method of Rickwood
VM., Rickwood D, Willson MT, Mitochondria, a Practical Approach, Oxford Wa
shington DC., IRL Press, 1987, pp.1-16).
That is, cells are suspended in a sucrose-based buffer and lysed using a glass homogenizer. The nuclei are pelleted, the mitochondrial fraction is further enriched and purified using a continuous Percoll gradient to separate defects from normal mitochondria and remove most cell debris. Scanning and transmission electron microscopy is used to calculate the purity and morphology of the mitochondrial fraction. Maintenance of membrane mitochondrial potential is analyzed by DePsipher dye as described above in Example 1 in combination with FACS analysis for a quick calculation of the total number and concentration of both functional and defective mitochondria present. Only the fractions containing more than 90% functional mitochondria are used for further studies. When matured oocytes ability FVB strain mice inhibits mitochondrial flag mentation in FVB strain mouse oocytes cultured in a) in vitro are cultured in vitro, very high rates of (-
75%) goes through natural fragmentation (Morita et al. Dev Biol. 1999)
Sep 1; 213 (1): 1-17). This model is used to test each mitochondrial enriched fraction for its ability to suppress oocyte fragmentation.
The ovulated oocytes were exfoliated from their cumulus cells and described by Van Blerkom et al. (Hum Repr
od. 1988 Oct; 13 (10): 2857-68), and the mitochondrial-enriched fraction is injected in a dose-response manner. Mature oocytes were calculated to contain approximately 100,000 mitochondria (Jansen and de Boer, Mol Cell Endocrinol. 1998 Oc.
t 25; 145 (1-2): 81-8). Inject between 2000 and 20,000 mitochondria in a volume of 5-15 picoL. A control group of oocytes is left intact or injected with the buffer used for mitochondrial suspension or the mitochondrial depleted fraction. Defective mitochondria from Percoll gradients are also injected to determine the possible deleterious effects of defective mitochondria on oocyte survival. All oocytes are then cultured and evaluated for fragmentation after 24 and 48 hours. This model is used to identify the optimal mitochondrial number and type for injection to suppress fragmentation.

As expected, mitochondria collected from stem cells are expected to be effective in suppressing fragmentation and have the advantage of strong replication ability. or injection of mitochondria from stem cells to b) normal mouse zygotes which had been fertilized in vitro to provide long-term suppression of the cell death? Increasing maternal age and in vitro fertilization resulted in an apoptosis rate of approximately 30% in mouse zygotes and in embryos compared to zygotes obtained from in vivo fertilized young mothers (approximately 2% fragmentation rate). Combine higher cell death index at the blastocyst stage (Jurisicova et al., Mol Hum Reprod. 1998 Feb; 4 (2): 139-
45). Furthermore, analysis of human blastocyst cell death rates showed that approximately 30% of embryos preferentially eliminate inner cell mass or activate cell death in most cells. To determine whether mitochondrial infusion can suppress zygote apoptosis and further provide suppression during late developmental stages, zygotes from aged mice (44 weeks old ICR strain) The pups are injected with a concentrated fraction of mitochondria and their development into the blastocyst stage is observed in vitro. The number of mitochondria infused is calculated using the method described in the previous experiment and the concentration fine-tuned if necessary. On day 4.5, the blastocyst number and cell death rate are recorded, paying particular attention to the inner cell mass.

Further studies examine the effect of mitochondrial injection on protection from cell death induced by various toxins as an artificial trigger for apoptosis. In particular, mitochondrial injection was shown to activate cell death pathways all during blastocyst formation (Bergeron et al., 1998 Gen. Dev. 12,1304-1314), hyperglycemia (Moley et al. al. Nat Med 1998 Dec; 4 (12): 1421-4) and whether it is possible to prevent apoptosis induced by treatment with DMBA. In these experiments, the appropriate mitochondrial-injected zygotes were cultured in KSOM medium until the early blastocyst stage was reached, at which time the experimental treatment was performed with doxorubicin (200 nM), glucose (20 mM) selective medium. Or DMBA (1μ
In vitro with either M). A zygote injected with a buffer or a mitochondrial deletion fraction that develops to the blastocyst stage is used as a control. 24
Blastocyst number and cell death index are determined as described above at time of toxin addition (Jurisicova et al., 1998, supra).

Expected results show that somatic mitochondria are diluted by subsequent cell division of the preimplantation embryo and are undetectable by the blastocyst stage (Ebert et al. 1989, J Reprod. Fertil
Jan; 82 (1): 145-9 9). Stem cell mitochondria are detectable at least 80 hours after injection into mouse oocytes Van Blerkom et al. (Hum Reprod.
1998 Oct; 13 (10): 2857-68), which must behave more similarly to oocyte mitochondria. Donor stem cell mitochondria are capable of replication,
When lasting to the blastocyst stage, protection from natural in vitro apoptosis and reduction of cell death rate after toxin administration must be observed. c) To determine whether injection of normal outgoing foster measuring mitochondrial mice born stem cells mitochondria from implanted zygote to jeopardize the span of normal development and life, as the in FVB zygote Various various stem cell or somatic cell mitochondrial concentrated fractions were injected and transplanted into pseudopregnant females. At least 20 in each group
Get offspring. Offspring are followed for 18 months for the detection of any developmental abnormalities, reproductive dysfunction, or reduced life span that can be attributed to the deleterious effects of donor mitochondrial infusion on prenatal and postnatal development. Furthermore, since 75% of the oocytes from this strain are normally apoptotic in vitro, the female progeny was further tested to determine whether the donor mitochondria were replicated to the progeny to generate heteroplasmy. To determine, the rate of fragmentation of those oocytes in vitro is calculated. All parameters are compared to progeny generated from sham injection zygotes.

Another method for determining donor stem cell mitochondrial replication capacity is strain C5.
7B16 / J and NZB / BINJ (Jackson laboratories) (Meirelles and
Smith, Genetics 1998 Feb; 148 (2): 877-83), as reported by mtDN.
The use of A restriction fragment length polymorphism (RFLP). The FVB strain is examined to determine if it contains RFLPs of mtDNA similar to either of the two strains, and based on these results, the TS or ES cell line is derived from the opposing strain. Mitochondrial-rich fractions from these genetically distinct cells are injected into FVB zygotes. The replicative capacity of the injected mitochondria can then be confirmed in its progeny by determining the RFLP status of the isolated mitochondria.

As expected, the progeny produced by mitochondrial injection of donor stem cells should have a normal lifespan and be phenotypically normal. If the donor mitochondria are replicable and capable of forming heteroplasmy, then these mice can have improved reproductive function and reduced oocyte apoptosis in vitro. The ability of heteroplasmy formation is important for the success of any future clinical studies aimed at correcting hereditary mitochondrial disease. D) No rescue of embryo fragmentation mediated by DNA deficiency. Both male and female gamete subsets contain defective DNA (Sun et al., Biol.
Reprod. 1997 Mar; 56 (3): 602-7, Lopes et al., Fertil Steril 1998 Mar; 69 (3): 52
8-32). Twigg et al. (Hum Reprod 1998 J due to ROS-induced sperm DNA loss)
ul; 13 (7): 1864-71) clearly show the ability of these spermatozoa to undergo decondensation and pronucleation, indicating that the early stages of embryonic development, even when the paternal DNA is fragmented. Suggest that there may be. Rescue of embryos with chromosomal abnormalities is not desirable. Genetic analysis of the cell death pathway of mouse gene cells can suppress apoptosis in the female genetic system when the trigger has no survival signal, but when the initiation factor is DNA deficient It suggests that apoptosis cannot be suppressed. Doerksen and Trasler (Biol Reprod 1996 Nov; 55 (5): 1155-) treated male mice with the drug 5-azacytidine (5-AZC), which interferes with DNA methylation and induces sperm DNA deficiency. Use the model developed by 62). Female mice mated with these treated males produce embryos with a high rate of fragmentation secondary to chromosomal defects and a low pregnancy rate (Doerksen and Trasler, 1996, supra). In this experiment, male animals were treated with 5-AZC (3 weeks, 4 mg / kg), sperm were collected from the cauda epididymis, and stem cells mitochondria or buffer were co-injected into mouse oocytes of the FVB strain. To do.

As expected, the failure of mitochondrial injection to suppress embryo fragmentation in this model confirms that human embryos whose cell death pathway was activated by DNA deficiency would not be rescued. Furthermore, Cohen and his collaborators (Lancet 1997 Jul
19; 350 (9072): 186-7) reported that cytoplasmic injection of donor oocytes into oocytes of 7 patients showed no improvement in embryo quality for two couples. Described.
These two couples were the only cases in which men suffered from severe minor asthenia gravis, which was shown to be associated with a high degree of sperm DNA deficiency (Sun et al., Biol Reprod 1
997 Mar; 56 (3): 602-7; Lopes et al., Fertil Steril 1998 Mar; 69 (3): 528-32, Hum
Reprod 1998 Mar; 13 (3): 703-8). The presence of DNA fragmentation in sperm may explain why the injection was unsuccessful in these two cases. Example 4 Overexpression of Mcl-1 and Bcl-xL in stem cell mitochondria to increase inhibition of cell death in mouse and human embryos Both mitochondrial localization in mouse and human oocytes and embryos 2 Species cell death suppressors, Bcl-xL and Mcl-1, are abundantly expressed. Varying levels of maternally stored transcripts were found for these two proteins in human oocytes, and variations in these proteins may lead to altered susceptibility to cell death triggers. It suggests something unknown.

The ubiquitous chicken b-actin promoter (pCAX-Hadjantonakis et
al., 1998, supra), forming transfected ES cells that overexpress Bcl-x _L or Mcl-1. Transfection systems were selected based on their resistance to neomycin and using Western blot analysis, Mcl-1 or B within their mitochondrial fractions.
Measured for protein levels of cl-x _L. Another mitochondrial-localized protein, cytochrome C, is used as a loading control to demonstrate high levels of Bcl-xL and Mcl-1 in the mitochondrial-rich fraction.
Upon establishing increased levels of protein expression on the mitochondrial membrane in these cells, mitochondria were isolated and used in experiments similar to those described above.
Thus, early embryos can be promoted with more functional mitochondria, but also with mitochondria containing higher protein content, either Bcl-x _L or Mcl-1.

As expected, if these transfected mitochondria are superior in suppressing cell death compared to their non-transfected equivalents, inhibition of apoptosis in this model and normal embryos Bcl-x _L or Mc in the development of
The significance of any of 1-1 will be established. Example 5 Injection of mitochondria into human oocytes during ICSI and rescue of fragmented embryos Two cycles of IVF followed by highly fragmented embryos (grade 4 or 5) or slow developing embryos (fertilization). 72 hours later produce only 6 cells or less) 2
0 patients will be collected for the pilot study. Women must first have normal day 3 serum FSH levels (<10 IU / L in our lab), but if the results of preliminary studies look promising, they should be higher than those with high serum FSH levels. Elderly women are also involved in the method. Ovulation induction is described in (Greenblatt et al., Fertil Ste
ril, 1995 Sep; 64 (3): 557-63), consisting of a long-term GnRH-agonist protocol with various human menopausal gonadotropins. The menstrual cycle is monitored using a combination of transvaginal ultrasound and serum estradiol measurements. Human ciliated gonadotropin is administered 36 hours before oocyte collection. Oocytes are transvaginally collected under the guidance of ultrasound. After oocyte harvest, it is removed by exposing the cumulus-oocyte complex to hyaluronidase in modified HTF medium. Each oocyte is measured with the first polar body present (MII) selected for maturity and ICSI. Immature oocytes are used for determination of mitochondrial function and mtDNA copy number and mutations as described in Example 2. Sperm is prepared as described above on the day of oocyte collection (Sun et al., Biol Reprod. 1997.
Mar; 56 (3): 602-7). The ICSI method used in this study was previously described in detail (Casper et al., 1996, supra). All submicroscopic injection methods are performed on the heated table of an inverted microscope (magnification x200 or x400). For microscopic injection, morphologically normal, motile sperm are selected from sperm / PVP droplets and fixed. Oocytes from each patient are divided into two groups. Group 1 oocytes are injected with one sperm as described above (Casper et al., Supra). The second group of oocytes was prepared from human cord blood-derived hematopoietic stem / progenitor cells prepared as described above.
Inject with one sperm aspirated in an injection pipette with between 000 and 20,000 normal mitochondria. The injection volume containing both sperm and mitochondria is maintained up to 15 picoL. After injection, cover oocytes with mineral oil and incubate at 37 ° C.
Of 100% of HTF medium supplemented with 5% human serum albumin in a plastic 60 × 15 mm Petri dish incubated in a humidified 5% CO ₂ environment. Cultured oocytes are measured 16-18 hours after ICSI for the presence of two pronuclei showing normal fertilization. Embryonic development and Veeck (1991; Acta Eur
Fertil, 1992 Nov-Dec; 23 (6): 275-88) is performed daily for evaluation. Evaluation of embryos (cell number x 1 / grade) was 48 and 72 hours for each embryo and 96 and 1
Determined for cell number at 20 hours. Expanded blastocysts that appear morphologically normal are transplanted 5 days after fertilization. If normal embryo development occurs in any of the control injected oocytes, they are transferred first. Pregnancy obtained with this method is closely followed and suggested to consider amniocentesis in order to exclude significant chromosomal abnormalities.
The babies born as a result of this method have had their cord blood collected and, if possible (ie, when mtDNA mutations were detected in non-fertilized oocytes) and in embryos first seen in these patients. Stored for the determination of mitochondrial heteroplasmy, which can be responsible for fragmentation or delayed development. Babies are also followed at birth and for as long as their parents agree, and occasionally thereafter by measuring normal development.

As expected, the first group of oocytes must result in embryos with delayed development or fully fragmented, consistent with the patient's history. In the second group of oocytes,
Injection of a dense fraction of stem cell mitochondria will allow intrauterine transplantation of some patients and normal development into the blastocyst stage with pregnancy.

Since these aspects are intended only as an example of one aspect of the invention, and all functionally equivalent aspects are within the scope of the invention, the invention is embodied in the invention described herein. The embodiment does not limit the range. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. These modifications are intended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are described as if each individual publication, patent or patent application was specifically and individually incorporated by reference in its entirety. As stated, it is incorporated by reference in its entirety to the same extent. All publications, patents and patent applications mentioned in this specification are
It is incorporated herein by reference for the purpose of describing and disclosing methodologies, etc. reported therein, which may be used in connection with the present invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

As used in the specification and the appended claims, the singular forms “a”, “an” and “the”
Includes multiple meanings unless the content clearly dictates otherwise. Thus, for example, "a gene" includes these multiple genes.

[Brief description of drawings]

[Figure 1]   FIG. 1 is a bar graph showing the effect of mitochondrial injection on preimplantation embryo development.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Kasper, Robert Ef             Ontario-M4 Canada             3bee7, Toronto, Meredei Skreset             Event 9 (72) Inventor Rogers, Ian             Canada, Ontario M5 R2             I1 Toronto Apartment 403 Bed             Dough Aether Road 25 (72) Inventor Taley, Jonathan             New Hampshire, United States             03087 Wyndham Stacy Circle 63 (72) Inventor Juri Shikova, Andrea             Canada / Ontario L4C4K             9. Litchimond Hill Traybone Dora             Eve 177 (72) Inventor Perez, Gloria Eye             02148 Mo, Masachi Yusetsu, United States             Ruden No. 610 Kennedy Dry             Bu 250 F-term (reference) 4B024 AA10 AA20 BA80 GA11 GA18                       GA30 HA20                 4B065 AA90X AA90Y AB10 AC20                       BA01 BA30 CA60

Claims (33)

[Claims] 1. A method for enhancing the developmental capacity of an egg cell or zygote, which method comprises increasing the intracellular amount of replicating mitochondria in the egg cell or zygote. 2. The method according to claim 1, wherein the intracellular amount of replicating mitochondria is increased by introducing replicating mitochondria derived from stem cells or immortalized cell lines. 3. The method according to claim 2, wherein the replicating mitochondria are introduced by microinjection or electrofusion. 4. The method according to claim 2 or 3, wherein the stem cells are genetically modified. 5. The method of claim 1, 2 or 3 wherein the replicative mitochondria comprise mitochondrial DNA without deletions or mutations. 6. Replicating mitochondria containing at least 60% of other cytoplasmic components.
, Preferably 75% and most preferably 90% free. 7. The method of claim 2, wherein the replicating mitochondria derived from the stem cell or immortalized cell line contains about 2,000-20,000 mitochondria. 8. The method according to claim 1, wherein the egg cells or zygotes are from sports, zoos, pets and farm animals. 9. The method according to claim 1, which enhances the developing ability of human egg cells. 10. An egg cell or zygote obtained by the method according to claim 1, which has an increased intracellular amount of mitochondria. 11. The method of claim 9, further comprising fertilizing an egg cell to obtain a zygote with an increased intracellular amount of replicating mitochondria. 12. A zygote having an increased intracellular amount of mitochondria, which is obtained from the method according to claim 11. 13. A composition comprising replicating mitochondria for enhancing the developmental capacity of egg cells and zygotes. 14. The composition of claim 13, wherein the replicating mitochondria are derived from stem cells or immortalized cell lines. 15. The composition of claim 13, wherein the replicating mitochondria are derived from differentiated mammalian cells. 16. Reducing the adverse effects of mitochondrial DNA mutations in individual progeny affected by mitochondrial DNA mutations, including introducing replicating mitochondria containing healthy mitochondria into egg cells or zygotes from the individual. How to let. 17. The method of claim 16, wherein the replicating mitochondria comprise mitochondrial DNA without deletions or mutations that result in impaired oxidative phosphorylation and clinical pathology associated with muscle or nervous tissue. 18. After in vitro fertilization or embryo transfer in a female mammal, comprising implanting an embryo derived from an egg cell or zygote containing an increased intracellular amount of replicative mitochondria in the female mammal. For improving the development of the embryo of the chick. 19. (a) A donor cell nucleus derived from a non-human mammalian donor cell and 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 donor cell nucleus. Replicating mitochondria from the same non-human mammal into an enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) culturing the nuclear transfer unit to obtain an embryo; A method of cloning a non-human mammal by nuclear transfer comprising (c) implanting the embryo in the uterus of a surrogate mother of the species and (d) allowing the embryo to develop in the cloned mammal. 20. (a) A donor cell nucleus derived from a non-human mammalian donor cell and 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 donor cell nucleus. Introducing replicating mitochondria from the same non-human mammal into an enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) culturing the nuclear transfer unit to obtain an embryo. A method of making a non-human mammalian viable embryo comprising: 21. (a) A donor cell nucleus derived from a non-human mammalian donor cell and preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably the donor. Replicating mitochondria from the same non-human mammal from which the cell nucleus is derived is introduced into an enucleated recipient egg cell of the same species as the donor cell to form a nuclear transfer unit, and (b) the nuclear transfer unit is 2-cell developed. (C) transferring the cultured nuclear translocation unit to a host non-human mammal of the same species to develop the nuclear translocation unit into a fetus. Method of fetal cloning. 22. The method of claim 21, wherein the fetus is allowed to develop into a child. 23. The method according to any one of claims 19 to 22, wherein the donor cell nucleus is from mesoderm, endoderm or ectoderm. 24. The method according to any one of claims 19 to 23, wherein the non-human mammal is cow, sheep, pig, horse, goat and buffalo. 25. The donor cell nucleus is an epithelial cell, nerve cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B-lymphocyte, T-lymphocyte, erythrocyte, macrophage, monocyte, fibroblast or muscle cell. 25. The method according to any one of claims 19 to 24, which is from 26. The donor cell nucleus according to claim 19, wherein the donor cell nucleus is from an organ selected from the group consisting of skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra. The method according to one. 27. The method according to any one of claims 19 to 26, wherein the recipient oocyte to be enucleated is matured in vitro or in vivo before enucleation. 28. The method according to any one of claims 19 to 27, wherein the enucleated recipient oocyte is an intermediate II stage oocyte. 29. The method according to any one of claims 19 to 28, wherein the donor nucleus is membrane-bound. 30. The method according to any one of claims 19 to 29, wherein the donor nucleus is a whole blastomere. 31. The donor nucleus is nucleoplasm aspirated from a blastomere.
The method according to any one of 0. 32. A recipient egg cell comprising a perivitelline space and a donor cell nucleus and replicating mitochondria deposited in the perivitelline space. 33. The recipient egg cell of claim 32, wherein the replicating mitochondria are derived from the same species and cell type as the donor cell nucleus, or from the same individual from which the donor cell nucleus is derived.