Background of the Invention [0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0003] Researchers have been developing methods for cloning mammalian animals over the past two decades. Some reported methods include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting that cell or a nucleus isolated from the cell into an enucleated oocyte (e. g., the nucleus of the oocyte was previously extracted), and (3) culturing the embryo in vivo.

[0004] The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in live offspring (s). McGrath & Solter, 1983, Science 220: 1300-1302. Subsequently, others described the production of chimeric murine embryos (e. g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGCs). These cells are and can give rise to pluripotent cells.

Matsui et al., 1992, Cell 70 : 841-847 and Resnick et al., 1992, Nature 359 : 550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13: 38. In 1998, researchers reported that murine cumulus cells can be used as nuclear donors in cloning techniques for establishing cloned murine animals. Wakayama et al., 1998, Nature 394 : 369-374.

[0005] Another nuclear transfer experiment was reported in 1986, where an ovine (sheep) embryonic cell was used as a nuclear donor in a cloning process that resulted in a cloned lamb. Willadsen, 1986, Nature 320: 63-65. More recently, other lambs were reported to be cloned from ovine embryonic cells; serum deprived somatic cells; cells isolated from embryonic discs; and somatic mammary tissue.

Campbell et al., 1996, Nature 380 : 64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 385 : 810-813; and PCT Publications WO 96/07732 and WO 97/07669. Other approaches for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668. Publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.

[0006] Yet another nuclear transfer experiment resulted in a cloned bovine animal (cattle), where the animal was cloned using an embryonic cell derived from a 2-64 cell embryo as a nuclear donor. This bovine animal was reportedly cloned by utilizing nuclear transfer techniques set forth in U. S. Patents 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed where an inner cell mass cell of a blastocyst stage embryo was utilized as a nuclear donor in a nuclear transfer procedure. Sims & First, 1993, Theriogenology 39: 313 and Keefer et al., 1994, Mol. Reprod. Dev. 38 : 264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized a PGC isolated from fetal tissue as a nuclear donor. Delhaise et al., 1995, Reprod.

Fert. Develop. 7 : 1217-1219; Lavoir 1994, J. Reprod. Dev. 37 : 413-424; and PCT application WO 95/10599 entitled"Embryonic Stem Cell-Like Cells."Additional nuclear transfer procedures are described in U. S. Patent Application Nos. 6,011,197, 6,107,543, and 5,945,577.

[0007] With regard to porcine animals (swine), researchers have reported methods for obtaining chimeric animals, and cloned animals. See., e. g., Prather et al., 1989, Biology of Reproduction 41 : 414-418 ; Piedrahita et al., 1998, Biology of

Reproduction 58 : 1321-1329; and WO 94/26884,"Embryonic Stem Cells for Making Chimeric and Transgenic Ungulates,"Wheeler, published November 24,1994. See also, U. S. Patent Application number 09/199, 138, entitled"Method of Cloning Porcine Animals", filed on November 24,1998, U. S. Provisional Patent Application No. 60/221,434, filed July 28,2000, entitled"Method of Cloning Porcine Animals." [0008] Also, researchers have reported nuclear transfer experiments using porcine nuclear donors and porcine oocytes. See., e. g., Nagashima et al., 1997, Mol.

Reprod. Dev. 48 : 339-343; Nagashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41 : 414-419; Prather et al., 1990, Exp. Zool. 255 : 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro. 259 : 257-266; Terlouw et al., 1992, Theriogenology 37 : 309, Pokajaeva et al., Nature 407,86-90 (2000); Onishi et al., Science 289 1188-1190 (2000); and Betthauser et al., Nature Biotechnology 18: 1055-1059 (2000).

[0009] Each reference in the foregoing section is hereby incorporated by reference in its entirety.

[00010] Despite the progress in producing mammalian animals using nuclear transfer methods, there remains a need in the art for methods for efficient production of transgenic mammalian animals by such procedures.

Summarv [00011] The invention relates in part to cloning technologies for mammalian animals. In particular, the materials and methods described herein relate to methods for producing transgenic animals by encapsulating exogenous nucleic acid molecules in a membrane that acts as an artificial nuclear membrane. These encapsulated nucleic acids are incorporated into nuclear transfer methods to deliver the exogenous nucleic acids in a manner that enhances their uptake and use by the recipient cell. Such methods can be used for the production of stem cells and for the production of cloned embryos, fetuses, and animals.

[00012] In a first aspect, the invention features a method of producing a transgenic mammalian embryo by nuclear transfer. Such an embryo can be referred to herein as a"cloned embryo."In this aspect, one or more pseudonuclei, and one or more nuclear donor cells, or nuclei thereof, are translocated into an enucleated oocyte. In an alternate aspect, one or more pseudonuclei are translocated into a nuclear donor cell prior to translocation of the nuclear donor cell into an enucleated oocyte. In either aspect, the result is a"cybrid"comprising the nuclear contents of the nuclear donor, together with the nucleic acids contained within the pseudonuclei.

[00013] The terms"pseudonuclei"and"pseudonucleus"as used herein refer to one or more exogenous nucleic acid molecules encapsulated within a nuclear membrane created in vitro. The concept of pseudonuclei as described herein is intended to include"pseudocells"or other artificial cells known to those of skill in the art. See, e. g., Chang, 1997,"Artificial cells and bioencapsulation in bioartificial organs,"Ann. N. Y. Acad. Sci. 831: 249-59.

[00014] In preferred embodiments, pseudonuclei are prepared by contacting one or more nucleic acid molecules with components obtained from amphibian egg extracts, preferably extracts obtained from Xenopus laevis eggs. Such extracts are described in Leno, G. H., 1998, Cell-free systems to study chromatin remodeling, Methods in Cell Biology 53,497-515; Lohka, M. J., 1998, Analysis of nuclear envelope assembly using extracts of Xenopus eggs, Methods in Cell Biology 53,367- 395; Newport, J., 1987, Nuclear reconstitution in vitro: stages of assembly around protein-free DNA, Cell 48,205-217 ; Laskey, R. A., Kearsey, S. E., and Mechali, M., 1985, Analysis of chromosome replication in eggs of Xenopus laevis, In :"Genetic Engineering: Principles and Methods"7,135-148; Leno, G. H. and Laskey, R. A., 1991, DNA replication in cell-free extracts from Xenopus laevis, Methods in Cell Biology 36,561-579.; Leno, G. H. and Laskey, R. A., 1991, The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts, J Cell Biol. 112, 557-566; Meier, J. Campbell, K. H., Ford, C. C., Stick, R., and Hutchison, C. J., 1991, The role of lamin LIII in nuclear assembly and DNA replication in cell-free

extracts of Xenopus eggs, J. Cell Sci. 98,271-279; each of which is hereby incorporated by reference in its entirety.

[00015] Such pseudonuclei can be prepared by encapsulating any nucleic acids.

In certain embodiments, the nucleic acids are DNA molecules, most preferably minichromosomes or artificial chromosomes. Both minichromosomes and artificial chromosomes are well known to the skilled artisan. See, e. g., Kereso et al., 1996, Chromsome Research 4: 226-239; Hollo et al., 1996, Chromosoyne Research 4: 240- 247; U. S. Patent No. 6,025,155; U. S. Patent No. 6,077,697; Cooke, H., 2001 Cloning and Stem Cells 3: 243-9; Saffery et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5705- 10; Kuroiwa, et al., 2002, Nat. Biotechnol. 10: 1086-90; each of which is hereby incorporated by reference in its entirety.

[00016] The pseudonuclei can also be further surrounded by an additional membrane that is equivalent to the plasma membrane of cells. Such surrounded pseudonuclei are described herein as"pseudocells."Pseudocells can function in all respects as pseudonuclei, and the methods described herein with respect to pseudonuclei also apply fully to pseudocells.

[000171 Amphibian eggs are typically obtained by natural or induced ovulation of a female amphibian. Such eggs are obtained from the animal in the metaphase II stage of meiosis, and are referred to herein as"non-activated"eggs. While the amphibian egg extract can be obtained from non-activated eggs, in certain preferred embodiments the amphibian egg extract is obtained from activated amphibian eggs.

Activation results in eggs that continue through meiosis beyond the metaphase II stage to interphase. Amphibian oocytes can be activated, for example, by increasing intracellular calcium with an ionophore such as A23187, and can be recognized by the contraction of the pigment in the animal hemisphere of the egg.

[00018] The skilled artisan will understand that, in addition to one or more components from an amphibian egg extract, additional material may be included to form pseudonuclei. For example, activated egg extract components may be combined with components obtained from other cell types, such as oocytes, non-activated eggs,

mammalian cells, etc., and contacted with the nucleic acid molecules to form the pseudonuclei.

[00019] Preferably, cloned embryos of the instant invention are produced by a nuclear transfer procedure using (a) a nuclear donor, (b) one or more pseudonuclei, and (c) an oocyte, where the oocyte is at a stage allowing formation of the embryo. In these procedures, one or more pseudonuclei are translocated into the oocyte, prior to, simultaneously with, or following translocation of the nuclear donor.

[00020] In preferred embodiments, (1) the oocyte is an enucleated oocyte; (2) the oocyte preferably originates from an ungulate animal; (3) the oocyte has been matured; (4) the oocyte has been matured for more than 14 hours; (5) the oocyte has been matured for more than 40 hours; (6) the nuclear donor is placed in the perivitelline space of the oocyte; (7) the nuclear donor utilized for nuclear transfer can arise from any mammalian cell type (e. g., a non-embryonic cell, a primordial germ cell, a genital ridge cell, a differentiated cell, a fetal cell, a non-fetal cell, a non-primordial germ cell, a cell isolated from an asynchronous population of cells, a cell isolated from a synchronous population of cells, a cell isolated from an existing animal, an embryonic stem cell, an embryonic germ cell, an amniotic cell, an allantoic cell, a chorionic cell, a cumulus cell, and a fetal fibroblast cell); (8) the nuclear transfer comprises the step of translocation of the nuclear donor and one or more pseudonuclei into the recipient oocyte; (9) the translocation can comprise the step of injection of the nuclear donor and/or the pseudonuclei into the recipient oocyte; (10) the translocation can comprise the step of fusion of the nuclear donor and or the pseudonuclei with the oocyte; (11) the fusion can comprise the step of delivering one or more electrical pulses to the nuclear donor and or the pseudonuclei and the oocyte; (12) the fusion can comprise the step of delivering a suitable concentration of at least one fusion agent to the nuclear donor and or the pseudonuclei and the oocyte; (13) the nuclear transfer may comprise the step of activation of the oocyte; (14) the activation is accomplished by (i) increasing intracellular levels of divalent cations in a cell, and (ii) reducing phosphorylation of cellular proteins in the cell; (15) the activation is accomplished by (i) introducing a divalent ion ionophore to

a cell, and (ii) introducing a protein kinase inhibitor to a cell; (16) the divalent ion ionophore is a Ca2+ ionophore ; (17) the Ca2+ ionophore is ionomycin; (18) the protein kinase inhibitor is DMAP; and (19) activation is accomplished by introducing DMAP and ionomycin to a cell.

[00021] The term"nuclear transfer"as used herein refers to introducing a full complement of nuclear DNA from one cell to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See., e. g., Nagashima et al., 1997, Mol. Reprod. Dev. 48 : 339-343; Nagashima et al., 1992, J.

Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41 : 414-419; Prather et al., 1990, Exp. Zool. 255 : 355-358; Saito et al., 1992, Assis. Reprod. Tech. Andro.

259 : 257-266; and Terlouw et al., 1992, Theriogenology 37 : 309, and each reference discussed in the Background of the Invention section of the instant application, each of which is incorporated herein by reference in its entirety including all figures, tables and drawings. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.

[00022] The term"nuclear donor"as used herein refers to a cell or a nucleus from a cell that is translocated into a nuclear acceptor. A nuclear donor may be a totipotent cell. In addition, a nuclear donor may be any cell type, including, but not limited to a non-embryonic cell, a non-fetal cell, a differentiated cell, a somatic cell, an embryonic cell, a fetal cell, an embryonic stem cell, a primordial germ cell, a genital ridge cell, a cumulus cell, an amniotic cell, an allantoic cell, a chorionic cell, a fetal fibroblast cell, a hepatocyte, an embryonic germ cell, an adult cell, a cell isolated from an asynchronous population of cells, and a cell isolated from a synchronized population of cells where the synchronous population is not arrested in the Go stage of the cell cycle. A nuclear donor cell can also be a cell that has differentiated from an embryonic stem cell. See, e. g., Piedrahita et al., 1998, Biol.

Reprod. 58 : 1321-1329; Shim et al., 1997, Biol. Reprod. 57 : 1089-1095 ; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28 : 173-189; and Wheeler, 1994, Reprod.

Fertil. Dev. 6 : 563-568, each of which is incorporated herein by reference in its

entirety including all figures, drawings, and tables. In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved.

[00023] A nuclear donor cell can itself be a transgenic cell prior to the nuclear transfer steps described herein using pseudonuclei. Such transgenic cells can be produced by the methods described herein.

[00024] The term"enucleated oocyte"as used herein refers to an oocyte which has had its nucleus removed. Typically, a needle can be placed into an oocyte and the nucleus can be aspirated into the needle. The needle can be removed from the oocyte without rupturing the plasma membrane. This enucleation technique is well known to a person of ordinary skill in the art. See, U. S. Patent 4,994,384; U. S. Patent 5,057,420; and Willadsen, 1986, Nature 320 : 63-65.. Oocytes to be enucleated can be obtained from gilts; that is, female pigs that are nulliparous, or from sows; that is, female pigs that are at least monoparous.

[00025] An enucleated oocyte is preferably prepared from an oocyte that has been matured for greater than or equal to 14 hours, for greater than or equal to 20 hours, for greater than or equal to 28 hours, for greater than or equal to 36 hours. In particularly preferred embodiments, an enucleated oocyte is prepared from an oocyte that has been matured for greater than or equal to 40 hours, up to about 96 hours.

[000261 The terms"maturation"and"matured"as used herein refers to a process in which an oocyte is incubated in a medium in vitro. Maturation media can contain multiple types of components, including hormones and growth factors. Time of maturation can be determined from the time that an oocyte is placed in a maturation medium to the time that the oocyte is subject to a manipulation (e. g., enucleation, nuclear transfer, fusion, and/or activation). Oocytes can be matured in multiple media well known to a person of ordinary skill in the art. See, e. g., Mattioli et al., 1989,77leriogenology 31 : 1201-1207; Jolliff & Prather, 1997, Biol. Reprod.

56: 544-548; Funahashi & Day, 1993, J. Reprod. Fert. 98 : 179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38 : 339-343; Abeydeera et al., 1998, Biol. Reprod. 58 : 213-218; Funahashi et al., 1997, Biol. Reprod. 57 : 49-53 ; and Sawai et al., 1997,

Biol. Reprod. 57 : 1-6, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Oocytes can be matured for any period of time. In particularly preferred embodiments, oocytes are matured for the times described in the preceeding paragraph.

[00027] An oocyte can also be matured in vivo. Time of maturation may be the time that an oocyte receives an appropriate stimulus to resume meiosis to the time that the oocyte is manipulated. Similar maturation periods described above for in vitro matured oocytes apply to in vivo matured oocytes.

[00028j A variety of oocytes can be selected for maturation. For example, oocytes can be isolated from a pre-pubertal animal or a peri-pubertal animal.

[00029] Nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. In addition, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes.

[00030] The term"cybrid"as used herein refers to an oocyte having a nuclear donor inserted within. The term"cybrid"refers to an oocyte having a nuclear donor that is translocated into the oocyte. A nuclear donor may be fused with an oocyte, and the term"cybrid"includes oocytes that are not fused with a nuclear donor.

[00031j The invention relates in part to cloned mammalian embryos established by nuclear transfer of a nuclear donor and an non-enucleated oocyte. A cloned embryo may be established where nuclear DNA from the donor cell replicates during cellular divisions while nuclear DNA from an oocyte does not replicate. See, e. g., Wagoner et al., 1996,"Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,"Theriogenology 46 : 279-284.

[00032] The term"another ungulate"as used herein refers to a situation where a nuclear donor originates from an ungulate of a different species, genera or family than the ungulate from which the recipient oocyte originates. For example, a porcine cell can be used as a nuclear donor, while a recipient oocyte can be isolated from a

domestic cow. This example is not meant to be limiting and any ungulate species/family combination of nuclear donors and recipient oocytes are foreseen by the invention.

[00033] The term"translocation"as used herein in reference to nuclear transfer refers to combining a nuclear donor and or one or more pseudonuclei and a recipient oocyte. Translocation may be performed by such techniques as fusion and/or direct injection, for example.

[00034] The term"injection"as used herein in reference to embryos, refers to perforation of an oocyte, or the perivitelline membrane of an oocyte, with a needle, and insertion of a nuclear donor and/or one or more pseudonuclei in the needle into the oocyte or perivteline space.

In preferred embodiments, a nuclear donor and/or one or more pseudonuclei may be injected into the cytoplasm of an oocyte. This direct injection approach is well known to a person of ordinary skill in the art, as indicated by publications already incorporated herein in reference to nuclear transfer. For a direct injection approach to nuclear transfer, a whole cell may be injected into an oocyte, or alternatively, a nucleus isolated from a cell may be injected into an oocyte. Such an isolated nucleus may be surrounded by nuclear membrane only, or the isolated nucleus may be surrounded by nuclear membrane and plasma membrane in any proportion. An oocyte may be pre-treated to enhance the strength of its plasma membrane, such as by incubating the oocyte in sucrose prior to injection of a nuclear donor.

[00035] A nuclear donor and/or one or more pseudonuclei can also be placed into the perivitelline space of an oocyte for translocation into the oocyte. Preferably, techniques for placing a nuclear donor into the perivitelline space of an enucleated oocyte is well known to a person of ordinary skill in the art, and is fully described in patents and references cited previously herein in reference to nuclear transfer.

[00036] The term"fusion"as used herein refers to combination of portions of lipid membranes corresponding to a nuclear donor and/or one or more pseudonuclei and a recipient oocyte. Lipid membranes can correspond to plasma membranes of

cells or nuclear membranes, for example. Fusion can occur with addition of a fusion stimulus between a nuclear donor and/or one or more pseudonuclei and recipient oocyte when they are placed adjacent to one another, or when a nuclear donor and/or one or more pseudonuclei are placed in the perivitelline space of a recipient oocyte, for example. These techniques for translocation are fully described in references cited previously herein in reference to nuclear transfer.

[00037] The term"electrical pulses"as used herein refers to subjecting a nuclear donor and/or one or more pseudonuclei and recipient oocyte to electric current. For nuclear transfer, a nuclear donor and/or one or more pseudonuclei and recipient oocyte can be aligned between electrodes and subjected to electrical current.

Electrical current can be alternating current or direct current. Electrical current can be delivered to cells for a variety of different times as one pulse or as multiple pulses.

Cells are typically cultured in a suitable medium for delivery of electrical pulses.

Examples of electrical pulse conditions utilized for nuclear transfer are described in references and patents previously cited herein in reference to nuclear transfer.

[00038] The term"fusion agent"as used herein refers to any compound or biological organism that can increase the probability that portions of plasma membranes or nuclear membranes from different cells will fuse when a nuclear donor and/or one or more pseudonuclei are placed adjacent to a recipient oocyte. In preferred embodiments fusion agents are selected from the group consisting of polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses, and Sendai virus. These examples are not meant to be limiting and other fusion agents known in the art are applicable and included herein.

[00039] The term"suitable concentration"as used herein in reference to fusion agents, refers to any concentration of a fusion agent that affords a measurable amount of fusion. Fusion can be measured by multiple techniques well known to a person of ordinary skill in the art, such as by utilizing a light microscope, dyes, and fluorescent lipids, for example.

[00040] The term"activation"in regard to mammalian oocytes refers to any materials and methods useful for stimulating a cell to divide before, during, and after a nuclear transfer step. The term"cell"as used in the previous sentence refers to an oocyte, a cybrid, a nuclear donor, and an early stage embryo. These types of cells may require stimulation in order to divide after nuclear transfer has occurred. The invention pertains to any activation materials and methods known to a person of ordinary skill in the art.

[00041] The skilled artisan will understand that the order of translocating the nuclear donor and one or more pseudonuclei can be varied relative to activation of the oocyte. For example, an oocyte may be activated prior to translocating both the nuclear donor and one or more pseudonuclei; after translocating the nuclear donor, but before translocating the pseudonuclei ; after translocating the pseudonuclei but before translocating the nuclear donor; or after translocating both the nuclear donor and the pseudonuclei.

[00042] Although electrical pulses are sometimes sufficient for stimulating activation of cells, other non-electrical means for activation are useful and are often necessary for proper activation of a cell. Chemical materials and methods useful for non-electrical activation are described below in other preferred embodiments of the invention. When two or more chemical components are introduced to a cell for activating the cell, the components can be added simultaneously or in steps.

[00043] Examples of electrical processes for activation are well known in the art. Researchers have also reported non-electrical processes for activation. See, e. g., Grocholová et al., 1997, J. Exp. Zoology 277 : 49-56; Schoenbeck et al., 1993, Theriogenology 40 : 257-266; Prather et al., 1989, Biology of Reproduction 41 : 414- 418; Prather et al., 1991, Molecular Reproduction and Development 28 : 405-409; Mattioli et al., 1991, Molecular Reproduction and Development 30: 109-125; Terlouw et al., 1992,77zeriogenology 37 : 309 ; Prochazka et al., 1992, J. Reprod.

Fert. 96 : 725-734 ; Funahashi et al., 1993, Molecular Reproduction and Development 36 : 361-367; Prather et al., Bio. Rep. Vol. 50 Sup 1 : 282 ; Nussbaum et al., 1995, Molecular Reproduction and Development 41 : 70-75; Funahashi et al., 1995, Zygote

3 : 273-281; Wang et al., 1997, Biology of Reproduction 56 : 1376-1382; Piedrahita et al., 1989, Biology of Reproduction 58 : 1321-1329; Macháty et al., 1997, Biology of Reproduction 57 : 85-91; and Macháty et al., 1995, Biology of Reproduction 52 : 753- 758.

[00044] Examples of components that are useful for non-electrical activation include ethanol; inositol trisphosphate (IPs) ; divalent ions (e. g., addition of Ca2+ and/or Sur") ; microtubule inhibitors (e. g., cytochalasin B); ionophores for divalent ions (e. g., the Ca2+ ionophore ionomycin); protein kinase inhibitors (e. g., 6- dimethylaminopurine (DMAP)); protein synthesis inhibitors (e. g., cycloheximide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); and thapsigargin. It is also known that temperature change and mechanical techniques are also useful for non-electrical activation. The invention includes any activation techniques known in the art. See, e. g., U. S. Patent No. 5,496,720, entitled"Parthenogenic Oocyte Activation,"issued on March 5,1996, Susko-Parrish et al., and Wakayama et al., 1998, Nature 394 : 369-374, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.

[00045] When ionomycin and DMAP are utilized for non-electrical activation, ionomycin and DMAP may be introduced to cells simultaneously or in a step-wise addition, the latter being a preferred mode as described herein. Preferred concentrations of ionomycin are 0.5 je, M to 100 LM ; particularly preferred concentrations are greater than or equal to 5 J, M, 7.5 pM, 10 pM, 12.5 1M, 15 p, M, 17.5 1M, 20 pM, 22.5 M, 25 aM, 30 1M, 35 pLM, 40 M, 50 M, 60 , M, 75 jj. M, and 100 p1M. Preferred concentrations of DMAP are 0.5 mM to 50 mM; particularly preferred are concentrations greater than or equal to 0.75 mM, 0.8 mM, 0. 9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM 2.2 mM, 2.3 mM 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM.

[00046] The amount of time that cells are exposed to ionomycin and/or DMAP can also be modified to provide additional control over the activation process.

Preferably, cells are exposed to ionomycin for between 1 minute and about 1 hour. In

preferred embodiments, cells are exposed to ionomycin for about 2 minutes, about 5 minutes, about 7.5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, and about 50 minutes. Also preferably, cells are exposed to DMAP for between about 1 hour and about 12 hours.

In preferred embodiments, cells are exposed to DMAP for about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, and about 11 hours.

[00047] Cells obtained from an embryo or fetus produced by the methods described herein can also be used in a subsequent nuclear transfer step. The term "subsequent nuclear transfer"as described herein is also referred to as a"re-cloning" step. Preferably, a re-cloning step can be utilized to enhance nuclear reprogramming during nuclear transfer, such that a product of nuclear transfer is a live born animal.

The number of subsequent nuclear transfer steps is discussed in greater detail hereafter. l00048] Any of the preferred embodiments related to the translocation, injection, fusion, and activation steps described previously herein can relate to any subsequent nuclear transfer step.

[00049] The term"inner cell mass"as used herein refers to cells that give rise to the embryo proper. Cells that line the outside of the inner cell mass are referred to as the trophoblast of the embryo. Methods for isolating inner cell mass cells from an embryo are well known to a person of ordinary skill in the art, as discussed previously. The term"pre-blastocyst"is well known in the art and is referred to previously.

[00050] The term"ovulated in vivo"as used herein refers to an oocyte that is isolated from an animal a certain number of hours after the animal exhibits characteristics that is associated with estrus or following injection of exogenous gonadatrophins known to induce ovulation. The characteristics of an animal in estrus are well known to a person of ordinary skill in the art, as described in references disclosed herein. See, e. g., Gordon, 1977,"Embryo transfer and associated

techniques in pigs (Gordon, ed.),"CAB International, Wallingford UK, pp. 60-76 and Kojima, 1998,"Embryo transfer,"Manual ofpig emblyo transfer procedures, National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp. 7-21, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.

[00051] In another aspect the invention relates to a cloned embryo produced by a process comprising the steps of (a) translocation of a nuclear donor and one or more pseudonuclei into an oocyte to establish a nuclear transfer oocyte; and (b) non- electrical activation of the nuclear transfer oocyte to establish the embryo.

[00052] In preferred embodiments, (1) the nuclear donor is a cultured cell and is selected from any of the cell types described herein; (2) the nuclear donor is a totipotent cell or is isolated from a totipotent cell; (3) the nuclear donor is any cell type discussed herein (e. g., embryonic germ cell, cumulus cell, amniotic cell, fibroblast cell); (4) the translocation comprises the step of fusion; and (5) the process comprises the step of culturing the embryo in vitro. Any other preferred embodiments discussed herein with respect to embryos, and especially with regard to activation, pertains to this aspect of the invention.

[00053] In another aspect the invention relates to a cloned embryo. The embryo is preferably produced by a method comprising the steps of nuclear transfer between: (a) a nuclear donor, where the nuclear donor is a totipotent cell; (b) one or more pseudonuclei ; and (c) an oocyte, where the oocyte is at a stage allowing formation of the embryo.

[00054] The term"totipotent"as used herein refers to a cell that gives rise to a live born animal. The term"totipotent"can also refer to a cell that gives rise to all of the cells in a particular animal. A totipotent cell can give rise to all of the cells of an animal when it is utilized in a procedure for developing an embryo from one or more nuclear transfer steps. Totipotent cells may also be used to generate incomplete animals such as those useful for organ harvesting, e. g., having genetic modifications to eliminate growth of an organ or appendage by manipulation of a homeotic gene.

[00055] The term"live born"as used herein preferably refers to an animal that exists ex utero. A"live born"animal may be an animal that is alive for at least one second from the time it exits the maternal host. A"live born"animal may not require the circulatory system of an in utero environment for survival. A"live born"animal may be an ambulatory animal. Such animals can include pre-and post-pubertal animals. As discussed previously, a live born animal may lack a portion of what exists in a normal animal of its kind.

[00056] The term"cultured"as used herein in reference to cells refers to one or more cells that are undergoing cell division or not undergoing cell division in an in vitro environment. An in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as suitable liquid media or agar, for example. Specific examples of suitable in vitro environments for cell cultures are described in Culture of Animal Cells : a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells : a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; and Animal Cells : culture and media, 1994, D. C. Darling, S. J.

MorganJohn Wiley and Sons, Ltd., each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings. Examples of preferred cell culture media include, but are not limited to, mem-a, Basal Medium Eagle (BME), CR12, Dulbecco's Modified Eagle's Medium (DME), Dulbecco's Minimum Essential Medium (DMEM), high glucose DMEM, Glasgow Minimum Essential Medium, Ham's F12, Iscove's Modified Dulbecco's Medium, Medium 199, M2, M16, RPMI 1640, commercial media such as Amniomax@ and and EpiLife keratinocyte medium (Sigma), and mixtures of the above. Such media may contain one or more supplements such as serum (e. g., fetal calf serum) and/or one or more growth factors and/or cytokines as described herein.

[00057] Cells may be cultured in suspension and/or in monolayers with one or more substantially similar cells. Cells may be cultured in suspension and/or in monolayers with a heterogeneous population of cells. The term"heterogeneous"as utilized in the previous sentence can relate to any cell characteristics, such as cell type

and cell cycle stage, for example. Cells may be cultured in suspension, cultured as monolayers attached to a solid support, and/or cultured on a layer of feeder cells, for example. The term"feeder cells"is defined hereafter. Furthermore, cells may be successfully cultured by plating the cells in conditions where they lack cell to cell contact. In particularly preferred embodiments, cells are cultured until they form a confluent culture. Preferably, cultured cells undergo cell division and are cultured for at least 5 days, more preferably for at least 10 days or 20 days, and most preferably for at least 30 days. Preferably, a significant number of cultured cells do not terminate while in culture. The terms"terminate"and"significant number are defined"hereafter. Nearly any type of cell can be placed in cell culture conditions.

Cultured cells can be utilized to establish a cell line.

[00058] The term"cell line"as used herein refers to cultured cells that can be passaged at least one time without terminating. The invention relates to cell lines that can be passaged at least 1,2,5,10,15,20,30,40,50,60,80,100, and 200 times.

Cell passaging is defined hereafter.

[00059] The term"suspension"as used herein refers to cell culture conditions in which cells are not attached to a solid support. Cells proliferating in suspension can be stirred while proliferating using apparatus well known to those skilled in the art.

[00060] The term"monolayer"as used herein refers to cells that are attached to a solid support while proliferating in suitable culture conditions. A small portion of cells proliferating in a monolayer under suitable growth conditions may be attached to cells in the monolayer but not to the solid support. Preferably less than 15 % of these cells are not attached to the solid support, more preferably less than 10% of these cells are not attached to the solid support, and most preferably less than 5 % of these cells are not attached to the solid support.

[00061] The term"plated"or"plating"as used herein in reference to cells refers to establishing cell cultures in vitro. For example, cells can be diluted in cell culture media and then added to a cell culture plate, dish, or flask. Cell culture plates

are commonly known to a person of ordinary skill in the art. Cells may be plated at a variety of concentrations and/or cell densities.

[00062] The term"cell plating"can also extend to the term"cell passaging." Cells of the invention can be passaged using cell culture techniques well known to those skilled in the art. The term"cell passaging"refers to a technique that involves the steps of (1) releasing cells from a solid support or substrate and disassociation of these cells, and (2) diluting the cells in media suitable for further cell proliferation.

Cell passaging may also refer to removing a portion of liquid medium containing cultured cells and adding liquid medium to the original culture vessel to dilute the cells and allow further cell proliferation. In addition, cells may also be added to a new culture vessel which has been supplemented with medium suitable for further cell proliferation. In preferred embodiments, cells are passaged by releasing cells from a surface using one or more proteases, e. g. Streptomyces griseus protease. Cells that are released can then be diluted and transferred to fresh culture containers. In particularly preferred embodiments, a protease treatment, while releasing some cells from a surface, leaves a subset of cells adherent to the surface. The released cells can be removed, and fresh medium can be provided to those cells that remained adherent, which are also referred to as having been passaged, as they are now more more "dilute"in number than before the protease treatment.

[00063] The term"proliferation"as used herein in reference to cells refers to a group of cells that can increase in number over a period of time.

[00064] The term"confluence"as used herein refers to a group of cells where a large percentage of cells are physically contacted with at least one other cell in that group. Confluence may also be defined as a group of cells that grow to a maximum cell density in the conditions provided. For example, if a group of cells can proliferate in a monolayer and they are placed in a culture vessel in a suitable growth medium, they are confluent when the monolayer has spread across a significant surface area of the culture vessel. The surface area covered by the cells preferably represents about 50% of the total surface area, more preferably represents about 70%

of the total surface area, and most preferably represents about 90 % of the total surface area.

[00065] The term"non-embryonic cell"as used herein refers to a cell that is not isolated from an embryo. Non-embryonic cells can be differentiated or non- differentiated. Non-embryonic cells refers to nearly any somatic cell, such as cells isolated from an ex utero animal. These examples are not meant to be limiting.

[00066] For the purposes of the present invention, the term"embryo"or "embryonic"as used herein refers to a developing cell mass that has not implanted into an uterine membrane of a maternal host. Hence, the term"embryo"as used herein refers to a fertilized oocyte, a cybrid (defined herein), a pre-blastocyst stage developing cell mass, a blastocyst, and/or any other developing cell mass that is at a stage of development prior to implantation into an uterine membrane of a maternal host. Embryos of the invention may not display a genital ridge. Hence, an "embryonic cell"is isolated from and/or has arisen from an embryo.

[000671 An embryo can represent multiple stages of cell development. For example, a one cell embryo can be referred to as a zygote, a solid spherical mass of cells resulting from a cleaved embryo can be referred to as a morula, and an embryo having a blastocoel can be referred to as a blastocyst.

[00068] The term"fetus"as used herein refers to a developing cell mass that has implanted into the uterine membrane of a maternal host. A fetus can include such defining features as a genital ridge, for example. A genital ridge is a feature easily identified by a person of ordinary skill in the art, and is a recognizable feature in fetuses of most animal species. The term"fetal cell"as used herein refers to any cell isolated from and/or has arisen from a fetus or derived from a fetus, including amniotic cells. The term"non-fetal cell"is a cell that is not derived or isolated from a fetus.

[00069] The term"parturition"as used herein refers to a time that a fetus is delivered from female recipient. A fetus can be delivered from a female recipient by abortion, c-section, or birth.

[00070] The term"primordial germ cell"as used herein refers to a diploid precursor cell capable of becoming a germ cell. Primordial germ cells can be isolated from any tissue in a developing cell mass, and are preferably isolated from genital ridge cells of a developing cell mass. A genital ridge is a section of a developing cell mass that is well-known to a person of ordinary skill in the art. See, e. g., Strelchenko, 1996, lkeriogenology 45 : 130-141 and Lavoir 1994, J. Reprod. Dev.

37 : 413-424.

[00071] The term"embryonic stem cell"as used herein refers to pluripotent cells isolated from an embryo that are maintained in in vitro cell culture. Embryonic stem cells may be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any stage of development, including blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells may have a rounded cell morphology and may grow in rounded cell clumps on feeder layers. Embryonic stem cells are well known to a person of ordinary skill in the art. See, e. g., WO 97/37009, entitled"Cultured Inner Cell Mass Cell-Lines Derived from Ungulate Embryos,"Stice and Golueke, published October 9,1997, and Yang & Anderson, 1992, Theriogenology 38 : 315-335, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. See, e. g., Piedrahita et al., 1998, Biol. Reprod. 58 : 1321-1329; Wianny et al., 1997, Biol. Reprod. 57 : 756-764; Moore & Piedrahita, 1997, In Vitro Cell Biol.

Anim. 33: 62-71; Moore, & Piedrahita, 1996, Mol. Reprod. Dev. 45 : 139-144; Wheeler, 1994, Reprod. Fert. Dev. 6: 563-568; Hochereau-de Reviers & Perreau, Reprod. Nutr. Dev. 33: 475-493; Strojek et al., 1990, Theriogenology 33 : 901-903; Piedrahita et al., 1990, Theriogenology 34 : 879-901; and Evans et al., 1990, Theriogenology 33: 125-129, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.

[00072] The term"differentiated cell"as used herein refers to a precursor cell that has developed from an unspecialized phenotype to a specialized phenotype. For example, embryonic cells can differentiate into an epithelial cell lining the intestine.

Materials and methods of the invention can reprogram differentiated cells into

totipotent cells. Differentiated cells can be isolated from a fetus or a live born animal, for example.

[00073] The term"undifferentiated cell"as used herein refers to a precursor cell that has an unspecialized phenotype and is capable of differentiating. An example of an undifferentiated cell is a stem cell.

[00074] The term"asynchronous population"as used herein refers to cells that are not arrested at any one stage of the cell cycle. Many cells can progress through the cell cycle and do not arrest at any one stage, while some cells can become arrested at one stage of the cell cycle for a period of time. Some known stages of the cell cycle are Gi, S, G, and M. An asynchronous population of cells is not manipulated to synchronize into any one or predominantly into any one of these phases. Cells can be arrested in the M stage of the cell cycle, for example, by utilizing multiple techniques known in the art, such as by colcemid exposure.

Examples of methods for arresting cells in one stage of a cell cycle are discussed in WO 97/07669, entitled"Quiescent Cell Populations for Nuclear Transfer,"hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Additionally, cells that reach confluence can become arrested in one stage of the cell cycle, typically Gi. See, e. g., Wieser et al., Oncogene 18: 277-81 (1999); Afrakhte et al., Cell Growth Differ. 9: 983-988 (1998); Pande et al., Cytometry 24: 55-63 (1996); Allday and Farrell, J. Virology 68: 3491-3498 (1994).

[00075] The terms"synchronous population"and"synchronizing"as used herein refers to a fraction of cells in a population that are within a same stage of the cell cycle. Preferably, about 50% of cells in a population of cells are arrested in one stage of the cell cycle, more preferably about 70 % of cells in a population of cells are arrested in one stage of the cell cycle, and most preferably about 90% of cells in a population of cells are arrested in one stage of the cell cycle. Cell cycle stage can be distinguished by relative cell size as well as by a variety of cell markers well known to a person of ordinary skill in the art. For example, cells can be distinguished by such markers by using flow cytometry techniques well known to a person of ordinary skill in the art. Alternatively, cells can be distinguished by size utilizing techniques

well known to a person of ordinary skill in the art, such as by the utilization of a light microscope and a micrometer, for example. In a preferred embodiment, cells are synchronized by arresting them (i. e., cells are not dividing) in a discreet stage of the cell cycle.

[00076] The terms"embryonic germ cell"and"EG cell"as used herein refers to a cultured cell that has a distinct flattened morphology and can grow within monolayers in culture. An EG cell may be distinct from a fibroblast cell. This EG cell morphology is to be contrasted with cells that have a spherical morphology and form multicellular clumps on feeder layers. Embryonic germ cells may not require the presence of feeder layers or presence of growth factors in cell culture conditions.

Embryonic germ cells may also grow with decreased doubling rates when these cells approach confluence on culture plates. Embryonic germ cells of the invention may be totipotent.

[00077] A preferred precursor cell for establishing an embryonic germ cell culture is a genital ridge cell from a fetus. Genital ridge cells are preferably isolated from fetuses where the fetus is between 20 days and parturition, between 30 days and 100 days, more preferably between 35 days and 70 days and between 40 days and 60 days, and most preferably about a 55 day fetus. An age of a fetus can be determined as described above. The term"about"with respect to fetuses refers to plus or minus five days. As described herein, EG cells may be physically isolated from a primary culture of cells, and these isolated EG cells may be utilized to establish a cell culture that eventually forms a homogenous or nearly homogenous cell line of EG cells.

[00078] The terms"morphology"and"cell morphology"as used herein refers to form, structure, and physical characteristics of cells. For example, one cell morphology is whether a cell is flat or round in appearance when cultured on a surface or in the presence of a layer of feeder cells. Many other cell morphologies are known to a person of ordinary skill in the art and are cell morphologies are readily identifiable using materials and methods well known to those skilled in the art. See, e. g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I.

Freshney (ed.), Wiley-Liss, Inc.

[00079] The term"cumulus cell"as used herein refers to any cultured or non- cultured cell that is isolated from cells and/or tissue surrounding an oocyte. Persons skilled in the art can readily identify a cumulus cell. Examples of methods for isolating and culturing cumulus cells are discussed in Damiani et al., 1996, Mol.

Reprod. Dev. 45 : 521-534; Long et al., 1994, J. Reprod. Fert. 102 : 361-369 ; and Wakayama et al., 1998, Nature 394 : 369-373, each of which is incorporated herein by reference in its entireties, including all figures, tables, and drawings.

[00080] The term"amniotic cell"as used herein refers to a cultured or non- cultured cell isolated from amniotic fluid or tissues in contact with amniotic fluid.

Persons skilled in the art can readily identify an amniotic cell. Examples of methods for isolating and culturing amniotic cells are discussed in Bellow et al., 1996, Theriogenology 45 : 225; Garcia & Salaheddine, 1997, Theriogenology 47 : 1003-1008; Leibo & Rail, 1990, Theriogenology 33 : 531-552; and Vos et al., 1990, Vet. Rec. 127 : 502-504, each of which is incorporated herein by reference in its entirety, including all figures tables and drawings.

[00081] The term"allantoic cell"as used herein refers to a cultured or non- cultured cell isolated from the allantois, a layer of fetal membranes associated with the chorion in mammals. Persons skilled in the art can readily identify an allantoic- cell.

[00082] The term"chorionic cell"as used herein refers to a cultured or non- cultured cell isolated from the chorion, a layer of fetal membranes associated with the placenta in mammals. Persons skilled in the art can readily identify a chorionic cell.

[00083] The term"fetal fibroblast cell"as used herein refers to any differentiated fetal cell having a fibroblast appearance. Fibroblasts can have a flattened and elongated appearance when cultured on culture media plates. Fetal fibroblast cells can also have a spindle-like morphology, density limited for growth, and can have a finite life span in culture of approximately fifty generations. In addition, fetal fibroblast cells may rigidly maintain a diploid chromosomal content and may generate type I collagen. For a description of fibroblast cells, see, e. g.,

Culture of Animal Cells : a manual of basic techniques (33d edition), 1994, R. I.

Freshney (ed), Wiley-Liss, Inc [00084] The term"adult cell"as used herein refers to any cell isolated from an adult animal. Such an adult cell can be isolated from any part of the animal, including, but not limited to, skin from an ear, skin from an abdominal region, kidney, liver, heart, follicle, and lung. Procedures are set forth herein for culturing such adult cells.

[000851 In preferred embodiments, (1) nuclear donor cells of the invention comprise modified nuclear DNA; (2) modified nuclear DNA includes a DNA sequence that encodes a recombinant product; (3) a recombinant product is a polypeptide; (4) a recombinant product is a ribozyme; (4) a recombinant product is expressed in a biological fluid or tissue; (5) a recombinant product confers or partially confers resistance to one or more diseases; (6) a recombinant product confers resistance or partially confers resistance to one or more parasites; (7) a modified nuclear DNA comprises at least one other DNA sequence that can function as a regulatory element; (8) a regulatory element is selected from the group consisting of promoter, enhancer, insulator, and repressor ; and (9) a regulatory element is selected from the group consisting of milk protein promoter, urine protein promoter, blood protein promoter, lacrimal duct protein promoter, synovial protein promoter, mandibular gland protein promoter, casein promoter, (3-casein promoter, melanocortin promoter, milk serum protein promoter, a-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, a-actin promoter.

[00086] The term"modified nuclear DNA"as used herein refers to a nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or animal of the invention that has been manipulated by one or more recombinant DNA techniques. Examples of recombinant DNA techniques are well known to a person of ordinary skill in the art, which can include (1) inserting a DNA sequence from another organism (e. g., a human organism) into target nuclear DNA, (2) deleting one or more DNA sequences from target nuclear DNA, and (3) introducing one or more base mutations (e. g., site- directed mutations) into target nuclear DNA. Cells with modified nuclear DNA can

be referred to as"transgenic cells"for the purposes of the invention. Transgenic cells can be useful as materials for nuclear transfer cloning techniques provided herein.

[00087] Particularly preferred are transgenic cells, embryos, fetuses, or animals in which one or more genes have been"knocked out."The term"knockout" as used herein refers to a cell, embryo, fetus, or animal in which a gene is functionally deleted; that is, in which a gene is no longer expressed in a functional manner. A gene can be functionally deleted by deletion or modification of the coding sequence for the gene. Preferred methods for producing a knockout are gene targeting strategies. In gene targeting, precise changes are inserted into specific locations of a host's DNA. For example, gene targeting constructs containing a modified gene of interest can be inserted into cells. The cells are cultured and screened for clones that contain homologous recombination events between the cellular genome and the gene targeting construct. The skilled artisan will understand that a diploid genome contains two alleles, each of which code for a gene of interest. For gene targeteting, one or both alleles may be functionally deleted to produce a"knockout"phenotype.

[00088] A gene can also be functionally deleted my masking the activity of the gene. For example, the gene for a-1, 3-galactosyltransferase can be masked by inserting a silencer sequence into the genome such that it prevents transcription of the gene. Such a gene may also be masked by inhibiting the activity of the gene product.

Alternatively, such a gene can be masked by removing the galactose moiety from polysaccharides that have been previously added by the gene product.

[00089] Methods and tools for insertion, deletion, and mutation of nuclear DNA of mammalian cells are well-known to a person of ordinary skill in the art. See, Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press; U. S. Patent 5,633,067,"Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,"DeBoer et al., issued May 27,1997; U. S. Patent 5,612,205,"Homologous Recombination in Mammalian Cells,"Kay et al., issued March 18,1997; and PCT publication WO 93/22432, "Method for Identifying Transgenic Pre-Implantation Embryos" ; WO 98/16630, Piedrahita & Bazer, published April 23,1998,"Methods for the Generation of

Primordial Germ Cells and Transgenic Animal Species,"each of which is incorporated herein by reference in its entirety, including all figures, drawings, and tables. These methods include techniques for transfecting cells with foreign DNA fragments and the proper design of the foreign DNA fragments such that they effect insertion, deletion, and/or mutation of the target DNA genome.

[00090] Any of the cell types defined herein can be altered to harbor modified nuclear DNA. For example, embryonic stem cells, embryonic germ cells, fetal cells, and any totipotent cell defined herein can be altered to harbor modified nuclear DNA.

[00091] Examples of methods for modifying a target DNA genome by insertion, deletion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, homologous recombination, gene targeting, transposable elements, and/or any other method for introducing foreign DNA. Other modification techniques well known to a person of ordinary skill in the art include deleting DNA sequences from a genome, and/or altering nuclear DNA sequences. Examples of techniques for altering nuclear DNA sequences are site-directed mutagenesis and polymerase chain reaction procedures. Therefore, the invention relates in part to cells that are simultaneously totipotent and transgenic. Such transgenic and totipotent cells can serve as nearly unlimited sources of donor cells for production of cloned transgenic animals.

[00092] The term"recombinant product"as used herein refers to the product produced from a DNA sequence that comprises at least a portion of the modified nuclear DNA. This product can be a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a polypeptide that binds to a regulatory element (a term described hereafter), a structural protein, an RNA molecule, and/or a ribozyme, for example. These products are well defined in the art. This list of products is for illustrative purposes only and the invention relates to other types of products.

[00093] The term"ribozyme"as used herein refers to ribonucleic acid molecules that can cleave other RNA molecules in specific regions. Ribozymes can bind to discrete regions on a RNA molecule, and then specifically cleave a region

within that binding region or adjacent to the binding region. Ribozyme techniques can thereby decrease the amount of polypeptide translated from formerly intact message RNA molecules. For specific descriptions of ribozymes, see U. S. Patent 5,354,855, entitled"RNA Ribozyme which Cleaves Substrate RNA without Formation of a Covalent Bond,"Cech et al., issued on October 11,1994, and U. S. Patent 5,591,610, entitled"RNA Ribozyme Polymerases, Dephosphorylases, Restriction Endoribonucleases and Methods,"Cech et al., issued on January 7,1997, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.

[00094] The terms"biological fluid"or"tissue"as used herein refers to any fluid or tissue in a biological organism. Fluids may include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, oviductal fluid, allantoic fluid, and synovial fluid. Tissues may include, but are not limited to, lung, heart, blood, liver, muscle, brain, pancreas, skin, and others.

[00095] The term"confers resistance"as used herein refers to the ability of a recombinant product to completely abrogate or partially alleviate the symptoms of a disease or parasitic condition. Hence, if a disease is related to inflammation, for example, a recombinant product can confer resistance to that inflammation if inflammation decreases upon expression of the recombinant product. A recombinant product may confer resistance or partially confer resistance to a disease or parasitic condition, for example, if a recombinant product is an anti-sense RNA molecule that specifically binds to an mRNA molecule encoding a polypeptide responsible for inflammation. Other examples of conferring resistance to diseases or parasites are described hereafter. In addition, examples of diseases are described hereafter.

[00096] Examples of parasites and strategies for conferring resistance to these parasites are described hereafter. These examples include, but are not limited to, worms, nematodes, insects, invertebrate, bacterial, viral, and eukaryotic parasites.

These parasites can lead to diseased states that can be controlled by materials and methods of the invention.

[000971 The term"regulatory element"as used herein refers to a DNA sequence that can increase or decrease an amount of product produced from another DNA sequence. A regulatory element can cause the constitutive production of the product (e. g., the product can be expressed constantly). Alternatively, a regulatory element can enhance or diminish production of a recombinant product in an inducible fashion (e. g., the product can be expressed in response to a specific signal). A regulatory element can be controlled, for example, by nutrition, by light, or by adding a substance to the transgenic organism's system. Examples of regulatory elements well-known to those of ordinary skill in the art are promoters, enhancers, insulators, and repressors. See, e. g., TransgenicAraimals, Generation and Use, 1997, Edited by L. M. Houdebine, Hardwood Academic Publishers, Australia, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings.

[00098] The term"promoters"or"promoter"as used herein refers to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably linked operatively to an adjacent DNA sequence. A promoter typically increases an amount of recombinant product expressed from a DNA sequence as compared to an amount of the expressed recombinant product when no promoter exists. A promoter from one organism specie can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism specie. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem.

Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art. Examples of promoter elements are described hereafter.

[00099] The term"enhancers"or"enhancer"as used herein refers to a DNA sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of a coding DNA sequence (e. g., a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer

element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream of a DNA sequence that encodes recombinant product. Enhancer elements can increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.

[000100] The term"insulators"or"insulator"as used herein refers to DNA sequences that flank the DNA sequence encoding the recombinant product. Insulator elements can direct recombinant product expression to specific tissues in an organism.

Multiple insulator elements are well known to persons of ordinary skill in the art.

See, e. g., Geyer, 1997, Curr. Opin. Genet. Dev. 7 : 242-248, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings.

[000101] The term"repressor"or"repressor element"as used herein refers to a DNA sequence located in proximity to the DNA sequence that encodes recombinant product, where a repressor sequence can decrease an amount of recombinant product expressed from that DNA sequence. Repressor elements can be controlled by binding of a specific molecule or specific molecules to a repressor element DNA sequence.

These molecules can either activate or deactivate a repressor element. Multiple repressor elements are available to a person of ordinary skill in the art.

[000102] The terms"milk protein promoter,""urine protein promoter,""blood protein promoter,""lacrimal duct protein promoter,""synovial protein promoter," and"mandibular gland protein promoter"refer to promoter elements that regulate the specific expression of proteins within the specified fluid or gland or cell type in an animal. For example, a milk protein promoter is a regulatory element that can control expression of a protein that is expressed in milk of an animal. Other promoters, such as casein promoter, a-lactalbumin promoter, whey acid protein promoter, uroplakin promoter, and a-actin promoter, for example, are well known to a person of ordinary skill in the art.

[000103] The term"cryopreserving"as used herein refers to freezing a cell, embryo, or animal of the invention. Cells, embryos, or portions of animals of the

invention are frozen at temperatures preferably lower than 0°C, more preferably lower than-80°C, and most preferably at temperatures lower than-196°C. Cells and embryos of the invention can be cryopreserved for an indefinite amount of time. It is known that biological materials can be cryopreserved for more than fifty years and still remain viable. For example, bovine semen that is cryopreserved for more than fifty years can be utilized to artificially inseminate a female bovine animal and result in the birth of a live offspring. Methods and tools for cryopreservation are well- known to those skilled in the art. See, e. g., U. S. Patent No. 5,160,312, entitled "Cryopreservation Process for Direct Transfer of Embryos,"issued to Voelkel on November 3,1992.

[000104] The term"thawing"as used herein refers to a process of increasing the temperature of a cryopreserved cell, embryo, or portions of animals. Methods of thawing cryopreserved materials such that they are active after a thawing process are well-known to those of ordinary skill in the art.

[000105] The terms"transfected"and"transfection"as used herein refer to methods of delivering exogenous DNA into a cell. These methods involve a variety of techniques, such as treating cells with high concentrations of salt, an electric field, liposomes, polycationic micelles, or detergent, to render a host cell outer membrane or wall permeable to nucleic acid molecules of interest. These specified methods are not limiting and the invention relates to any transformation technique well known to a person of ordinary skill in the art. See, e. g., Molecular Cloning, a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press and Transgenic Animals, Generation and Use, 1997, Edited by L.

M. Houdebine, Hardwood Academic Publishers, Australia, both of which were previously incorporated by reference.

[0001061 The terms"foreign DNA"as used herein refers to DNA that can be transfected into a target cell, where foreign DNA harbors at least one base pair modification as compared to the nuclear DNA of the target organism. Foreign DNA and transfection can be further understood and defined in conjunction with the term "modified nuclear DNA,"described previously.

[000107] The term"dissociating"as used herein refers to materials and methods useful for separating a cell away from another cell, where the cells originally contacted one another. For example, a blastomere (i. e., a cellular member of a morula stage embryo) can be pulled away from the rest of a developing cell mass by techniques and apparatus well known to a person of ordinary skill in the art. See, e. g., U. S. Patent 4,994,384, entitled"Multiplying Bovine Embryos,"issued on February 19,1991, hereby incorporated herein by reference in its entirety, including all figures, tables, and drawings. Alternatively, cells proliferating in culture can be separated from one another to facilitate such processes as cell passaging and formation of EG cells, which are described herein. In addition, dissociation of a cultured cell from a group of cultured cells can be useful as a first step in a process of nuclear transfer, as described hereafter. When a cell is dissociated from an embryo, a dissociation can be useful for such processes as re-cloning, a process described herein, as well as a step for multiplying a number of embryos.

[000108] The term"exogenous nucleic acid"as used herein refers to a nucleic acid molecule that is inserted into a cell, and that is from a source other than the cell into which the molecule has been inserted. In preferred embodiments, an exogenous nucleic acid is inserted into a cybrid, [000109] The term"cloned"as used herein refers to a cell, embryonic cell, fetal cell, and/or animal cell having a nuclear DNA sequence that is substantially similar or identical to a nuclear DNA sequence of another cell, embryonic cell, fetal cell, and/or animal cell. The terms"substantially similar"and"identical"are described herein. A cloned embryo can arise from one nuclear transfer process, or alternatively, a cloned embryo can arise from a cloning process that includes at least one re-cloning step. If a cloned embryo arises from a cloning procedure that includes at least one re-cloning step, then the cloned embryo can indirectly arise from a totipotent cell since the re- cloning step can utilize embryonic cells isolated from an embryo that arose from a totipotent cell.

[0001101 In preferred embodiments (1) the cloned embryo can be one member of a plurality of embryos, where the plurality of embryos share a substantially similar

nuclear DNA sequence; (2) the cloned embryo can be one member of a plurality of embryos, and the plurality of embryos can have an identical nuclear DNA sequence; (3) the cloned embryo has a nuclear DNA sequence that is substantially similar to a nuclear DNA sequence of a live born animal; (4) one or more cells of the cloned embryo have modified nuclear DNA; (5) the cloned embryo is subject to manipulation; (6) the manipulation comprises the step of culturing the embryo in a suitable medium; (7) the medium can comprise feeder cells; (8) the manipulation of an embryo comprises the step of implanting the embryo into reproductive tract of a female animal ; (9) the female animal is preferably an ungulate animal; (10) the estrus cycle of the female is not synchronized with the development cycle of the embryo; (11) the estrus cycle of the female is synchronized with the development cycle of the embryo; and (12) the manipulation comprises the step of incubating the embryo in an artificial environment.

[000111] The term"substantially similar"as used herein in reference to nuclear DNA sequences refers to two nuclear DNA sequences that are nearly identical. Two sequences may differ by copy error differences that normally occur during replication of nuclear DNA. Substantially similar DNA sequences are preferably greater than 97 % identical, more preferably greater than 98 % identical, and most preferably greater than 99% identical. The term"identity"as used herein can also refer to to amino acid sequences. It is preferred and expected that nuclear DNA sequences are identical for cloned animals. Examples of methods for determining whether cloned animals and cells from which they are cloned have substantially similar or identical nuclear DNA sequences are microsatellite analysis and DNA fingerprinting analysis.

Ashworth et al., 1998, Nature 394 : 329 and Signer et al., 1998, Nature 394 : 329.

[000112] The term"plurality"as used herein in reference to embryos refers to a set of embryos having a substantially similar nuclear DNA sequence. In preferred embodiments, a plurality consists of 5 or more embryos, 10 or more embryos, 15 or more embryos, 20 or more embryos, 25 or more embryos, 30 or more embryos, 40 or more embryos, 50 or more embryos, 60 or more embryos, 70 or more embryos, 80 or more embryos, 90 or more embryos, 100 or more embryos, 200 or more

embryos, 300 or more embryos, 500 or more embryos, and 1000 or more embryos.

A plurality of embryos can also refer to a set of embryos that do not have substantially similar nuclear DNA sequences.

[000113] The term"culturing"as used herein with respect to embryos refers to laboratory procedures that involve placing an embryo in a culture medium. An embryo can be placed in a culture medium for an appropriate amount of time to allow stasis of an embryo, or to allow the embryo to grow in the medium. Culture media suitable for culturing embryos are well-known to those skilled in the art. See, e. g., Nagashima et al., 1997, Mol. Reprod. Dev. 48 : 339-343; Petters & Wells, 1993, J.

Reprod. Fert. (Suppl) 48 : 61-73; Reed et al., 1992, Theriogenology 37 : 95-109; Dobrinsky et al., 1996, Biol. Reprod. 55 : 1069-1074; U. S. Patent No. 5,213,979, First et al.,"In Vitro Culture of Bovine Embryos,"May 25,1993; U. S. Patent No.

5,096,822, Rosenkrans, Jr. et al.,"Bovine Embryo Medium,"March 17,1992, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.

[000114] The term"suitable medium"as used herein refers to any medium that allows cell proliferation or allows stasis of an embryo. If a medium allows cell proliferation, a suitable medium need not promote maximum proliferation, only measurable cell proliferation. A suitable medium for embryo development can be an embryo culture medium described herein by example. The term"feeder cells"is defined previously herein. Embryos of the invention can be cultured in media with or without feeder cells. In other preferred embodiments, the feeder cells can be cumulus cells or follicular cells.

[000115] The term"implanting"as used herein in reference to embryos refers to impregnating a female animal with an embryo described herein. Implanting techniques are well known to a person of ordinary skill in the art. See, e. g., Polge & Day, 1982,"Embryo transplantation and preservation,"Control of Pig Reproduction, DJA Cole and GR Foxcroft, eds., London, UK, Butterworths, pp. 227-291; Gordon, 1997,"Embryo transfer and associated techniques in pigs,"Controlled reproduction in pigs (Gordon, ed.), CAB International, Wallingford UK, pp. 164-182 ; and

Kojima, 1998,"Embryo transfer,"Manual of pig embryo transfer procedures, National Livestock Breeding Center, Japanese Society for Development of Swine Technology, pp. 76-79, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. Preferably, a plurality of embryos, as defined above, are transferred to a female animal to establish a pregnancy.

[000116] In establishing a pregnancy, embryo (s) are preferably transferred directly into the oviduct or uterus of the recipient maternal animal. In preferred embodiments, the embryos are transferred into the oviduct infundibulum, oviduct ampulla, oviduct isthmus, uterotubal junction, uterine horn, or uterine body. Most preferably, a specific location is selected for transfer, depending on the age/developmental stage of the embryo (s). For example, 1-to 3-cell embryos may be transferred into the oviduct, while embryos of 4+ cells are transferred into the uterus, while 3-or 4-cell embryos are transferred either into the oviduct or the uterus.

The embryo (s) may be allowed to develop in utero, or alternatively, the fetus may be removed from the uterine environment before parturition.

[000117] In particularly preferred embodiments, embryos having 1 cell, embryos having up to 2 cells, embryos having up to 3 cells, embryos having up to 4 cells, embryos having up to 5 cells, embryos having up to 7 cells, embryos having up to 10 cells, embryos having up to 15 cells, embryos having up to 20 cells, embryos having up to 30 cells, embryos having up to 40 cells, embryos having up to 50 cells, embryos having up to 75 cells, embryos having up to 100 cells, embryos having up to 200 cells, embryos having up to 300 cells, and embryos having up to 400 cells are transferred into the oviduct, most preferably into a region of the oviduct selected from the group consisting of the oviduct infundibulum, the oviduct ampulla, the oviduct isthmus, and the uterotubal junction.

[000118] In other particularly preferred embodiments, embryos having the cell numbers described above are transferred into the uterus, most preferably into a region of the uterus selected from the group consisting of the uterotubal junction, the uterine horn, and the uterine body.

[000119] In other particularly preferred embodiments, embryos activated for the times described above are transferred into the uterus, most preferably into a region of the uterus selected from the group consisting of the uterotubal junction, the uterine horn, and the uterine body.

[000120] The term"synchronized"as used herein in reference to estrus cycle, refers to assisted reproductive techniques well known to a person of ordinary skill in the art. Typically, estrogen and progesterone hormones are utilized to synchronize the estrus cycle of the female animal with the developmental stage of the embryo, although a female animal that has naturally gone into standing estrus can be used for this purpose. For example, for porcines, a recipient maternal animal and an embryo to be implanted in the recipient are said to be"synchronized"or"synchronous"when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs about 44 to 46 hours after the onset of standing estrus in the maternal recipient. The term "about"in this context refers to +/-0. 5 hours.

[000121] The term"developmental stage"as used herein refers to embryos of the invention and morphological and biochemical changes during embryo development. This developmental process is predictable for embryos from ungulates, and can be synchronized with the estrus cycle of a recipient animal.

[000122] In other preferred embodiments, one or more embryos are "asynchronous"with the recipient maternal animal. Preferably, a recipient maternal animal and an embryo to be implanted in the recipient are said to be"asynchronous" when the embryo is more developed than would be expected if the embryo and the maternal recipient were synchronized. For example, in porcines, when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs prior to the onset of standing estrus in the maternal recipient, and up to about 43 hours after the onset of standing estrus in the maternal recipient, the recipient maternal animal and the embryo are said to be"asynchronous."The term"about"in this context refers to +/- 0.5 hours. The skilled artisan will understand that this time period does not include

any time that an embryo may be stored in an inactive state between activation and implantation. For example, an embryo may be activated several days, or even months, before the onset of standing estrus in a recipient animal, and then frozen.

[000123] A recipient maternal animal and an embryo to be implanted in the recipient are also said to be"asynchronous"when the embryo is less developed than would be expected if the embryo and the maternal recipient were synchronized. For example, when either fertilization (for a sexually reproduced embryo, including one produced by artificial insemination) or activation (for a nuclear transfer embryo) occurs later than about 47 hours after the onset of standing estrus in the maternal recipient, the recipient maternal animal and the embryo are said to be "asynchronous."The term"about"in this context refers to +/-0.5 hours. The skilled artisan will understand that this time period does not include any time that an embryo may be stored in an inactive state between activation and implantation. For example, an embryo may be activated several days, or even months, before the onset of standing estrus in a recipient animal, and then frozen.

[000124] The term"artificial environment"refers to one that promotes viability or development of an embryo or other developing cell mass. An artificial environment can be a uterine environment or an oviductal environment of a species different from that of a developing cell mass. For example, a developing bovine embryo can be placed into an uterus or oviduct of an ovine animal. Stice & Keefer, 1993,"Multiple generational bovine embryo cloning,"Biology of Reproduction 48 : 715-719. Alternatively, an artificial development environment can be assembled in vitro. This type of artificial uterine environment can be synthesized using biological and chemical components known in the art.

[000125] In another aspect, the invention features a cloned fetus arising from an embryo of the invention. A fetus may be isolated from an uterus of a pregnant female animal and may be isolated from another part of a pregnant female animal in the case of an ectopic pregnancy. In this aspect a cloned fetus is prepared by a process comprising the steps of (a) preparation of a cloned embryo defined previously, and (b) manipulation of the cloned embryo such that it develops into a fetus.

[0001261 In other aspects, the invention features (2) a method for preparing a cloned fetus comprising the steps of (a) preparation of a cloned embryo defined previously, and (b) manipulation of the cloned embryo such that it develops into a fetus; (3) a method of using a cloned fetus of the invention comprising the step of isolating at least one cell type from a fetus (e. g., for establishing a cell line or for a subsequent nuclear transfer step); and (4) a method of using a cloned fetus of the invention comprising the step of separating at least one part of a fetus into individual cells (e. g., for establishing a cell line or for a subsequent nuclear transfer step).

[000127] In another aspect the invention features a cloned animal arising from an embryo of the invention. In other aspects, the invention relates to a cloned animal, where the animal is one member of a plurality of animals, and where the plurality of animals have a substantially similar nuclear DNA sequence. The term"substantially similar"in relation to nuclear DNA sequences is defined previously herein.

In preferred embodiments, (1) the plurality consists of five or more animals, ten or more animals, one-hundred or more animals, and one-thousand or more animals; and (2) the plurality of animals can have an identical nuclear DNA sequence.

The term"identical"in reference to nuclear DNA sequences is described previously herein.

[000128] In yet another aspect, the invention features a method of using a cloned animal, comprising the step of isolating at least one component from the animal.

[000129] The term"component"as used herein can relate to any portion of a animal. A component can be selected from the group consisting of fluid, biological fluid, cell, tissue, organ, gamete, embryo, and fetus. For example, precursor cells, as defined previously, may arise from fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses isolated from cloned organisms of the invention.

[000130] The term"gamete"as used herein refers to any cell participating, directly or indirectly, to the reproductive system of an animal. A gamete can be a specialized product from the gonads of an organism, where the gamete may transfer genetic material while participating in fertilization. Examples of gametes are

spermatocytes, spermatogonia, oocytes, and oogonia. Gametes can be present in fluids, tissues, and organs collected from animals (e. g., sperm is present in semen).

The invention relates to collection of any type of gamete from an animal. For example, methods of collecting semen and oocytes are known to a person of ordinary skill in the art. See, e. g., Gordon, 1997,"Introduction to controlled breeding in pigs, Embryo transfer and associated techniques in pigs,"Controlled reproduction in pigs (Gordon, ed.), CAB International, Wallingford UK, pp. 1-59 ; Mattioli et al., 1989, Theriogenology 31 : 1207-1207; Funahashi & Day, 1993, J. Reprod. Fert. 98 179- 185; Funahashi et al., 1997, Biol. Reprod. 57 : 49-53; Abeydeera et al., 1998, Biol.

Reprod. 58 : 213-218 ; and Sawai et al., 1997, Biol. Reprod. 57 : 1-6, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.

[000131] The term"tissue"is defined previously. The term"organ"relates to any organ isolated from an animal or any portion of an organ. Examples of organs and tissues are neuronal tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary and kidney. These examples are not limiting and the invention relates to any organ and any tissue isolated from a cloned animal of the invention.

[000132] In a preferred embodiments, the invention relates to (1) fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses can be subject to manipulation; (2) the manipulation can comprise the step of cryopreserving the gametes, embryos, and/or fetal tissues; (3) the manipulation can comprise the step of thawing the cryopreserved items; (4) the manipulation can comprise the step of separating the semen into X-chromosome bearing semen and Y-chromosome bearing semen; (5) the manipulation comprises methods of preparing the semen for artificial insemination; (6) the manipulation comprises the step of purification of a desired polypeptide (s) from the biological fluid or tissue; (7) the manipulation comprises concentration of the biological fluids or tissues; (8) the manipulation can comprise the step of transferring one or more fluids, cloned cells, cloned tissues, cloned organs, and/or portions of cloned organs to a recipient organism (e. g., the recipient organism

may be of a different specie than the donor source); (9) the recipient organism is non- human; and (10) the recipient organism is human.

[000133] The term"separating"as used herein in reference to separating semen refers to methods well known to a person skilled in the art for fractionating a semen sample into sex-specific fractions. This type of separation can be accomplished by using flow cytometers that are commercially available. Methods of utilizing flow cytometers from separating sperm by genetic content are well known in the art. In addition, semen can be separated by its sex-associated characteristics by other methods well known to a person of ordinary skill in the art. See, U. S. Patents 5,439,362,5,346,990, and 5,021,244, entitled"Sex-Associated Membrane Proteins and Methods for Increasing the Probability that Offspring Will Be of a Desired Sex," Spaulding, issued on August 8,1995, September 13,1994, and June 4,1991 respectively, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.

[000134] The term"purification"as used herein refers to increasing the specific activity of a particular polypeptide or polypeptides in a sample. Specific activity can be expressed as a ratio between the activity or amount of a target polypeptide and the concentration of total polypeptide in the sample. Activity can be catalytic activity and/or binding activity, for example. Also, specific activity can be expressed as a ratio between the concentration of target polypeptide and the concentration of total polypeptide. Purification methods include dialysis, centrifugation, and column chromatography techniques, which are well-known procedures to a person of ordinary skill in the art. See, e. g., Young et al., 1997,"Production of biopharmaceutical proteins in the milk of transgenic dairy animals,"BioPharm 10 (6): 34-38.

[000135] The term"transferring"as used herein can relate to shifting a group of cells, tissues, organs, and/or portions of organs to an animal. Cells, tissues, organs, and/or portions of organs can be, for example, (a) developed ira vitro and then transferred to an animal, (b) removed from a cloned animal and transferred to another animal of a different species, (c) removed from a cloned animal and transferred to another animal of the same species, (d) removed from one portion of an animal (e. g.,

cells from a leg of an animal) and then transferred to another portion of the same animal (e. g., a brain of the same animal), and/or (e) any combination of the foregoing.

[000136] The term"transferring"as used herein refers to adding fluids, cells, tissues, and/or organs to an animal and refers to removing cells, tissues, and/or organs from an animal and replacing them with cells, tissues, and/or organs from another source. For example, neuronal tissue from a cloned organism can be grafted into an appropriate area in the nervous system of a human to treat neurological diseases (e. g., Alzheimer's disease). In another example, a heart or part of a heart may be removed from a cloned animal and can be surgically inserted into a human from which a heart or part of the heart was previously removed. Surgical methods for accomplishing this preferred aspect of the invention are known to a person of ordinary skill in the surgical arts. Transferring procedures may include the step of removing cells, tissues, fluids and/or organs from a recipient organism before a transfer step.

[000137] In other aspects the invention features (1) a cloned animal prepared by a process comprising the steps of: (a) preparation of a cloned embryo by any one of the methods described herein for producing such a cloned embryo, and (b) manipulation of the cloned embryo such that it develops into a live born animal; and (2) a process for cloning a animal, comprising the steps of: (a) preparation of a cloned embryo by any one of the methods described herein for preparing such a cloned embryo, and (b) manipulation of the cloned embryo such that it develops into a live born animal.

[000138] In preferred embodiments (1) the manipulation can comprise the step of implanting the embryo into a uterus of an animal; (2) the estrus cycle of the animal can be synchronized to the developmental stage of the embryo; and (3) the manipulation can comprise the step of implanting the embryo into an artificial environment.

[000139] In yet another aspect, the present invention relates to transgenic stem cells and stem cell cultures. In various embodiments, such stem cells and cultures may be prepared by a process comprising (a) preparation of a cloned embryo by any one of the methods described herein for producing such a cloned embryo, and (b) culturing one or more cells obtained from said embryo to generate said cultures.

Alternatively, stem cells and cultures may be obtained by transfer of one or more pseudonuclei into an oocyte, followed by parthenogenic activation of said oocyte.

Detailed Description Of The Preferred Embodiments [000140] Preparation of Pseudonuclei [000141] Amphibian eggs, and particularly eggs of the frog Xenopus laevis, have proved to be excellent sources of cell-free systems for studying chromatin remodeling and assembly of the cell nucleus. Systems have been developed that assemble nucleosomes and nuclear envelopes on purified DNA. The development of these cell-free systems has been facilitated by earlier microinjection studies. Thus, the behavior of DNA templates injected into eggs has been well documented and these studies have defined a performance target for the development of cell-free systems that mimic microinjected cells. A crucial feature that allows such efficient cell-free systems is the extraordinarily rapid rate of cell division cycles following fertilization.

The first division occurs 90 minutes after fertilization, and the next 11 cell cycles last only ~ 30 minutes each. To sustain these remarkable rates, the egg is provided with a stockpile of macromolecules and organelles for chromatin remodeling and nuclear assembly. This is the key advantage of Xe710pus eggs and egg extracts.

[000142] Nucleic acid molecules, such as mammalian artificial chromosomes, can be incubated in Xenopus egg extract for various lengths of time and monitored for nuclear envelope assembly by treatment with the lipophilic dye, Nile red. Complete envelope assembly can be confirmed by probing the chromosomes with an antibody that recognizes lamin LIII, a structural protein that accumulates within a functional nucleus. The DNA content of these artificial chromosome-containing"pseudonuclei"

are then determined by staining with a DNA specific dye, e. g., Hoechst 33258, and quantitated by fluorescence microscopy.

[000143] The size of nuclei can be determined in this fashion, and the DNA content of the pseudonuclei (e. g., 1 artificial chromosome, 2 artificial chromosomes, etc.) can be selected for microinjection into oocytes. A donor nucleus and the enveloped artificial chromosome (s) can be microinjected together into an enucleated oocyte arrested in metaphase of meiosis II, resulting in nuclear envelope breakdown, chromosome condensation, and alignment of all chromosomes on the metaphase plate.

Enclosure of the nucleic acid molecule (e. g., artificial chromosome) within a nuclear envelope in the pseudonucleus results in the reorganization and stabilization of chromatin and facilitates the association of the artificial chromosome with the chromosomes from the donor nucleus on the metaphase plate. Once aligned on the plate, all chromosomes, including the artificial chromosome, segregates appropriately resulting in the appearance of the artificial chromosome in all cells of the cloned embryo.

[000144] Nuclear Transfer [0001451 Nuclear transfer (NT) techniques are well known to a person of ordinary skill in the art. See, e. g., U. S. Patent No. 4,664,097,"Nuclear Transplantation in the Mammalian Embryo by Microsurgery and Cell Fusion,"issued May 12,1987, McGrath & Solter U. S. Patent 4,994,384 (Prather et al.) ; 5,057,420 (Massey et al.) ; 5,945,577 (Stice et al.) ; and 6,011,197 (Strelchenko et al.), each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings.

[000146] Nuclear Donors [000147] For NT techniques, a donor cell may be separated from a growing cell mass, isolated from a primary cell culture, or isolated from a cell line. The entire cell may be placed in the perivitelline space of a recipient oocyte or may be directly injected into the recipient oocyte by aspirating the nuclear donor into a needle, placing the needle into the recipient oocyte, releasing the nuclear donor and removing

the needle without significantly disrupting the plasma membrane of the oocyte. Also, a nucleus (e. g., karyoplast) may be isolated from a nuclear donor and placed into the perivitelline space of a recipient oocyte or may be injected directly into a recipient oocyte, for example.

[000148] Recipient Oocytes [000149] A recipient oocyte is typically an oocyte with a portion of its ooplasm removed, where the removed ooplasm comprises the oocyte nucleus. Enucleation techniques are well known to a person of ordinary skill in the art. See e. g., Nagashima et al., 1997, Mol. Reprod. Dev. 48 : 339-343; Nagashima et al., 1992, J.

Reprod. Dev. 38 : 37-78; Prather et al., 1989, Biol. Reprod. 41 : 414-418; Prather et al., 1990, J. Exp. Zool. 255 : 355-358; Saito et al., 1992, Assis. Reprod. Tech.

Andro. 259 : 257-266; and Terlouw et al., 1992, Theriogenology 37 : 309, each of which is incorporated herein by reference in its entirety including all figures, tables, and drawings.

[000150] Oocytes can be isolated from either oviducts and/or ovaries of live animals by oviductal recovery procedures or transvaginal oocyte recovery procedures well known in the art and described herein. Furthermore, oocytes can be isolated from deceased animals. For example, ovaries can be obtained from abattoirs and oocytes can be aspirated from these ovaries. The oocytes can also be isolated from the ovaries of a recently sacrificed animal or when the ovary has been frozen and/or thawed.

[000151] Oocytes can be matured in a variety of media well known to a person of ordinary skill in the art. One example of such a medium suitable for maturing oocytes is depicted in an exemplary embodiment described hereafter. Oocytes can be successfully matured in this type of medium within an environment comprising 5 % C02 at 39°C. Oocytes may be cryopreserved and then thawed before placing the oocytes in maturation medium. Cryopreservation procedures for cells and embryos are well known in the art as discussed herein.

[000152] Components of an oocyte maturation medium can include molecules that arrest oocyte maturation. Examples of such components are 6- dimethylaminopurine (DMAP) and isobutylmethylxanthine (IBMX). IBMX has been reported to reversibly arrest oocytes, but the efficiencies of arrest maintenance are quite low. See, e. g., Rose-Hellkant and Bavister, 1996, Mol. Reprod. Develop. 44 : 241-249. However, oocytes may be arrested at the germinal vesicle stage with a relatively high efficiency by incubating oocytes at 31°C in an effective concentration of IBMX. Preferably, oocytes are incubated the entire time that oocytes are collected.

Concentrations of IBMX suitable for arresting oocyte maturation are 0.01 mM to 20 mM IBMX, preferably 0.05 mM to 10 mM IBMX, and more preferably about 0.1 mM IBMX to about 0.5 mM IBMX, and most preferably 0.1 mM IBMX to 0.5 mM IBMX. In certain embodiments, oocytes can be matured in a culture environment having a low oxygen concentration, such as 5% 02, 5-10% C02, and 85-90% N2.

[000153] A nuclear donor cell and a recipient oocyte can arise from the same species or different species. For example, a totipotent porcine cell can be inserted into a porcine enucleated oocyte. Alternatively, a totipotent wild boar cell can be inserted into a domesticated porcine oocyte. Any nuclear donor/recipient oocyte combinations are envisioned by the invention. Preferably the nuclear donor and recipient oocyte from the same specie. Cross-species NT techniques can be utilized to produce cloned animals that are endangered or extinct.

[000154] Oocytes can be activated by electrical and/or non-electrical means before, during, and/or after a nuclear donor is introduced to recipient oocyte. For example, an oocyte can be placed in a medium containing one or more components suitable for non-electrical activation prior to fusion with a nuclear donor. Also, a cybrid can be placed in a medium containing one or more components suitable for non-electrical activation. Activation processes are discussed in greater detail hereafter.

[000155]Injection/Fusion [000156] A nuclear donor and/or one or more pseudonuclei can be translocated into an oocyte using a variety of materials and methods that are well known to a person of ordinary skill in the art. In one example, a nuclear donor and/or one or more pseudonuclei may be directly injected into a recipient oocyte. This direct injection can be accomplished by gently pulling a nuclear donor into a needle, piercing a recipient oocyte with that needle, releasing the nuclear donor into the oocyte, and removing the needle from the oocyte without significantly disrupting its membrane. Appropriate needles can be fashioned from glass capillary tubes, as defined in the art and specifically by publications incorporated herein by reference.

[000157] In another example, at least a portion of plasma membrane from a nuclear donor and recipient oocyte can be fused together by utilizing techniques well known to a person of ordinary skill in the art. See, Willadsen, 1986, Nature 320: 63- 65, hereby incorporated herein by reference in its entirety including all figures, tables, and drawings. Typically, lipid membranes can be fused together by electrical and chemical means, as defined previously and in other publications incorporated herein by reference. Thus, in a similar fashion, the nuclear membrane surrounding a pseudonucleus (or the"plasma membrane"surrounding a pseudocell) can be fused with either a recipient oocyte or nuclear donor cell.

[000158] Examples of non-electrical means of cell fusion involve incubating the cells to be fused in solutions comprising polyethylene glycol (PEG), and/or Sendai virus. PEG molecules of a wide range of molecular weight can be utilized for cell fusion.

[000159] Processes for fusion that are not explicitly discussed herein can be determined without undue experimentation. For example, modifications to cell fusion techniques can be monitored for their efficiency by viewing the degree of cell fusion under a microscope. The resulting embryo can then be cloned and identified as a totipotent embryo by the same methods as those previously described herein for

identifying totipotent cells, which can include tests for selectable markers and/or tests for developing an animal.

[000160] Activation [000161] Methods of activating oocytes and cybrids are known to those of ordinary skill in the art. See, U. S. Patent 5,496,720,"Parthenogenic Oocyte Activation,"Susko-Parrish et al., issued on March 5,1996, hereby incorporated by reference herein in its entirety including all figures, tables, and drawings.

[000162] Both electrical and non-electrical processes can be used for activating cells (e. g., oocytes and cybrids). Although use of a non-electrical means for activation is not always necessary, non-electrical activation can enhance the developmental potential of cybrids, particularly when young oocytes are utilized as recipients.

[000163] Examples of electrical techniques for activating cells are well known in the art. See, WO 98/16630, published on April 23,1998, Piedraheidra and Blazer, hereby incorporated herein in its entirety including all figures, tables, and drawings, and U. S. Patents 4,994,384 and 5,057,420. Non-electrical means for activating cells can include any method known in the art that increases the probability of cell division. Examples of non-electrical means for activating a nuclear donor and/or recipient can be accomplished by introducing cells to ethanol; inositol trisphosphate (IPs) ; Ca2+ ionophore and protein kinase inhibitors such as 6-dimethylaminopurine ; temperature change; protein synthesis inhibitors (e. g., cycloheximide); phorbol esters such as phorbol 12-myristate 13-acetate (PMA); mechanical techniques, thapsigargin, and sperm factors. Sperm factors can include any component of a sperm that enhance the probability for cell division. Other non-electrical methods for activation include subjecting the cell or cells to cold shock and/or mechanical stress.

[000164] Examples of preferred protein kinase inhibitors are protein kinase A, G, and C inhibitors such as 6-dimethylaminopurine (DMAP), staurosporin, 2- aminopurine, sphingosine. Tyrosine kinase inhibitors may also be utilized to activate cells.

[0001651 Activation materials and methods that are not explicitly discussed herein can be identified by modifying the specified conditions defined in the exemplary protocols described hereafter and in U. S. Patent No. 5,496,720.

[000166] Activation efficiency and totipotency that result from any modifications of activation procedures can be identified by methods described previously in the section entitled"Identification of Totipotent Cells."Methods for identifying totipotent embryos can include one or more tests, such as (a) identifying specific markers for totipotent cells in embryos, and (b) by determining whether the embryos are totipotent by allowing them to develop into an animal. Therefore, the invention relates to any modifications to the activation procedures described herein even though these modifications may not be explicitly stated herein.

[000167] Activation methods may also be used to parthenogenically activate nucleated oocytes, in order to produce embryonic stem cells from the resulting "embryo."See, e. g. Birger et al., Proc. Natl. Acad. Sci. USA, 1996,93 (13): 6371-6 ; Park et al., Jpn. J. Vet. Res., 1998,46 (1) : 19-28.

[000168] Manipulation of Embryos Resulting from Nuclear Transfer [000169] An embryo resulting from a NT process can be manipulated in a variety of manners. The invention relates to cloned embryos that arise from at least one NT. Exemplary embodiments of the invention demonstrate that two or more NT procedures may enhance the efficiency for the production of totipotent embryos.

Exemplary embodiments indicate that incorporating two or more NT procedures into methods for producing cloned totipotent embryos may enhance placental development. In addition, increasing the number of NT cycles involved in a process for producing totipotent embryos may represent a necessary factor for converting non-totipotent cells into totipotent cells. An effect of incorporating two or more NT cycles upon totipotency of resulting embryos is a surprising result, which was not previously identified or explored in the art.

[000170] Incorporating two or more NT cycles into methods for cloned totipotent embryos can provide further advantages. Incorporating multiple NT

procedures into methods for establishing cloned totipotent embryos provides a method for multiplying the number of cloned totipotent embryos.

[000171] When multiple NT procedures are utilized for the formation of a cloned totipotent embryo, oocytes that have been matured for any period of time can be utilized as recipients in the first, second or subsequent NT procedures. For example, if a first NT and then a second NT are performed, the first NT can utilize an oocyte that has been matured for about 24 hours as a recipient and the second NT may utilize an oocyte that has been matured for less than about 36 hours as a recipient. Alternatively, the first NT may utilize an oocyte that has been matured for about 36 hours as a recipient and the second NT may utilize an oocyte that has been matured for greater than about 24 hours as a recipient for a two-cycle NT regime. In addition, both NT cycles may utilize oocytes that have been matured for about the same number of hours as recipients in a two-cycle NT regime.

[000172] For NT techniques that incorporate two or more NT cycles, one or more of the NT cycles may be preceded, followed, and/or carried out simultaneously with an activation step. As defined previously herein, an activation step may be accomplished by electrical and/or non-electrical means as defined herein. Exemplified embodiments described hereafter describe NT techniques that incorporate an activation step after one NT cycle. However, an activation step may also be carried out at the same time as a NT cycle (e. g., simultaneously with the NT cycle) and/or an activation step may be carried out prior to a NT cycle. Cloned totipotent embryos resulting from a NT cycle can be (1) disaggregated or (2) allowed to develop further.

[000173] If embryos are disaggregated, disaggregated embryonic derived cells can be utilized to establish cultured cells. Any type of embryonic cell can be utilized to establish cultured cells. These cultured cells are sometimes referred to as embryonic stem cells or embryonic stem-like cells in the scientific literature. The embryonic stem cells can be derived from early embryos, morulae, and blastocyst stage embryos. Multiple methods are known to a person of ordinary skill in the art for producing cultured embryonic cells. These methods are enumerated in specific references previously incorporated by reference herein.

[000174] If embryos are allowed to develop into a fetus in utero, cells isolated from that developing fetus can be utilized to establish cultured cells. In preferred embodiments, primordial germ cells, genital ridge cells, and fetal fibroblast cells can be isolated from such a fetus. Cultured cells having a particular morphology that is described herein can be referred to as embryonic germ cells (EG cells). These cultured cells can be established by utilizing culture methods well known to a person of ordinary skill in the art. Such methods are enumerated in publications previously incorporated herein by reference and are discussed herein. In particularly preferred embodiments, Streptomyces griseus protease can be used to remove unwanted cells from the embryonic germ cell culture.

[000175] Cloned totipotent embryos resulting from NT can also be manipulated by cryopreserving and/or thawing the embryos. See, e. g., Nagashima et al., 1989, Japanese J. Anim. Reprod. 35 : 130-134 and Feng et al., 1991, Theriogenology 35 : 199, each of which is incorporated herein by reference in its entirety including all tables, figures, and drawings. Other embryo manipulation methods include in vitro culture processes; performing embryo transfer into a maternal recipient; disaggregating blastomeres for NT processes; disaggregating blastomeres or inner cell mass cells for establishing cell lines for use in NT procedures; embryo splitting procedures; embryo aggregating procedures; embryo sexing procedures; and embryo biopsying procedures. The exemplary manipulation procedures are not meant to be limiting and the invention relates to any embryo manipulation procedure known to a person of ordinary skill in the art.

[000176] Culture of Embryos In Vitro [000177] Cloning procedures discussed herein provide an advantage of culturing cells and embryos in vitro prior to implantation into a recipient female. Methods for culturing embryos in vitro are well known to those skilled in the art. See, e. g., Nagashima et al., 1997, Mol. Reprod. Dev. 48 : 339-343; Petters & Wells, 1993, J.

Reprod. Fert. (Suppl) 48 : 61-73 ; Reed et al., 1992, Theriogenology 37 : 95-109 ; and Dobrinsky et al., 1996, Biol. Reprod. 55 : 1069-1074, each of which is incorporated herein by reference in its entirety, including all figures, tables, and drawings. In

addition, exemplary embodiments for media suitable for culturing cloned embryos in vitro are described hereafter. Feeder cell layers may or may not be utilized for culturing cloned embryos in vitro. Feeder cells are described previously and in exemplary embodiments hereafter.

[000178] Developnzent of Embryos In Utero [000179] Cloned embryos can be cultured in an artificial or natural uterine environment after NT procedures and embryo in vitro culture processes. Examples of artificial development environments are being developed and some are known to those skilled in the art. Components of the artificial environment can be modified, for example, by altering the amount of a component or components and by monitoring the growth rate of an embryo.

[000180] Methods for implanting embryos into the uterus of an animal are also well known in the art, as discussed previously. Preferably, the developmental stage of the embryo (s) is correlated with the estrus cycle of the animal.

[000181] Embryos from one specie can be placed into the uterine environment of an animal from another specie. For example it has been shown in the art that bovine embryos can develop in the oviducts of sheep. Stice & Keefer, 1993,"Multiple generational bovine embryo cloning,"Biology of Reproduction 48 : 715-719. The invention relates to any combination of a embryo in any other ungulate uterine environment. A cross-species in utero development regime can allow for efficient production of cloned animals of an endangered species. For example, a wild boar embryo can develop in the uterus of a domestic porcine sow.

[000182] Once an embryo is placed into the uterus of a recipient female, the embryo can develop to term. Alternatively, an embryo can be allowed to develop in the uterus and then can be removed at a chosen time. Surgical methods are well known in the art for removing fetuses from uteri before they are born.

[000183] Examples : [000184] Example 1: Preparation of Egg Extracts [000185] Sexually mature Xenopus laevis females (Nasco Co., Fort Atkinson, WI); are obtained and allowed to acclimate to the laboratory environment for several weeks. Unfertilized eggs are obtained by priming a frog for ovulation by injection of 100 IU pregnant mare serum gonadotropin (PMSG) 4 days before egg collection.

This results in follicle cell stimulation and oocyte growth. Twelve to fifteen hours before egg collection, each primed frog is injected in the dorsal lymph sac with 500 IU human chronic gonadotropin (HCG) to induce ovulation. Injected frogs are then placed within individual tanks containing a salt solution that prevents spontaneous activation of the eggs (e. g., high-salt Barth's solution: 100 mM NaCI, 15 mM Tris- HCI, pH 7.4,2mM KCI, 2 mM NaHCOs, 1 mM MgS04, 0.5 mM Na2HP04) (Blow and Laskey, 1986). After egg laying slows or stops, the frogs are returned to water and allowed to recover for a minimum of 3 months.

[000186] Eggs from each frog are transferred in high-salt Barth's, and the jelly coat of the egg is removed by incubation in 2% cysteine-HCl, pH 7.8-7.9, for approximately 10 minutes at room temperature. Complete removal of the jelly coat results in the eggs"settling"together, i. e., no visible space is observed between adjacent eggs. Dejellied eggs are then rinsed with Barth's solution (88 mM NaCl, 15 mM Tris-HC1, pH 7.6,2 mM KC1, lmM MgCl2, 0.5 mM CaC12) (Blow and Sleeman, 1990) until debris is absent from the rinse solution. Discolored or misshapen eggs are removed.

[000187] Dejellied eggs are artificially activated by incubation of the eggs with calcium ionophore A23187 for approximately 5 min (e. g., 02-0.5 Fg/ml in a rinse solution) (Blow and Laskey, 1986; Newport, 1987). Following activation, eggs are rinsed extensively in ice-cold extraction buffer (50 mM HEPES/KOH, pH 7.4,50 mM KC1, 5 mM MgClz, 2 mM 2-mercaploethanol, 3, ug/ml leupeptin, 10, ug/ml cytochalasin B) (Blow and Laskey, 1986). After the final rinse in extraction buffer, eggs are packed by low-speed centrifugation (e. g., 1500 rpm for 2 min in an SW50.1

rotor (Beckman) at 4°C) (Blow and Sleeman, 1990). Excess buffer is removed from the surface of the eggs to prevent excessive dilution of the extract.

[000188] To separate the various egg components, packed eggs are centrifuged at 9000-10,000 g for 10-15 min in an SW50. lTi or SW60 Ti rotor (Beckman) at 2- 4°C (Blow and Laseky 1986; Hutchison et al., 1987, 1988; Blow and Sleeman, 1990). Egg components separate into three distinct fractions, including (1) a lipid cap, (2) a golden cytoplasmic layer, and (3) a plug of yolk platelets and pigment.

The cytoplasmic layer can be collected by puncturing the side of the tube with a syringe or by breaking through the lipid cap with a cooled pasteur pipet.

Cytochalasin B, which prevents actin polyumerization, is then added to this extract to a final concentration of 10 pg/mL. The extract is often contaminated with yolk, pigment, and lipid material, which can be cleared by centrifugation at 27,000-30,000 g for 10 to 15 min at 2-4°C.

[000189] This extract may be fractionated further by higher speed centrifugation.

Separation of the soluble components from the larger membrane vesicles can be achieved by high-speed centrifugation at 100,000xg for 1 hr in an SW50. lTi or SW60 Ti rotor (Beckman) at 4°C. Three distinct extract fractions are produced: (1) a transparent soluble fraction or"high-speed supernatant" (HSS), (2) a membranous or vesicular fraction, and (3) a golden, hard ribosomal pellet (Fig. 1).

[000190] In an alternative approach for the preparation of Xenopus extracts, dejellied eggs are crushed by centrifugation at 9000 g for 30 min at 4°C in modified extraction medium (20 mM HEPES, pH 7.5,70 mM KC1, 1 mM dithiothreitol, 5 % sucrose, 10 pg/ml leupeptin). The resultant supernatant is then centrifuged at 150,000 g for 60 min to produce an extract, which is used immediately. A more detailed description of this protocol has been published (Wolffe and Schild, 1991), which is hereby incorporated by reference.

[000191] Example 2: Preparing Pseudonuclei [000192] Mammalian artificial chromosomes, are incubated in the foregoing Xenopus egg extract for various lengths of time and monitored for nuclear envelope

assembly by treatment with the lipophilic dye, Nile red. Complete envelope assembly is confirmed by probing the chromosomes with an antibody that recognizes lamin LIII, a structural protein that accumulates within a functional nucleus.

[000193] Example 3. Nuclear Transfer [000194] A pseudonucleus containing a single artificial chromosome and a donor nucleus obtained from a cell isolated from a confluent culture are each drawn into a microinjection needle, and the needle placed against an enucleated recipient oocyte.

With controlled negative pressure, the oocyte membrane is drawn into the needle until the membrane ruptures. The pseudonucleus, donor cell nucleus, and any oocyte cytoplasm drawn into the needle is then expelled into the oocyte interior.

[000195] Oocytes/cybrids are activated by incubation in 5-15, uM calcium ionomycin (Calbiochem, San Diego, CA) for 5-20 minutes followed by incubation with 1.9 mM 6-dimethylaminopurine (DMAP) in CR2 for 3-4 hours. These concentrations and times can vary, depending on the species of interest. After DMAP incubation, cybrids are washed through two 35 mm plates containing TL-HEPES, and cultured in CR2 medium containing BSA (3 mg/ml) for 48 hours, then placed in NCSU 23 medium containing 0.4 % BSA for 24 hours followed by a final culture in NCSU 23 containing 10% FBS.

[000196] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.

[000197] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The cell lines, embryos, animals, and processes and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Modifications therein and other uses will occur to those skilled in the art. These

modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[000198] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[000199] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[000200] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms"comprising","consisting essentially of'and"consisting of"may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[000201] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described.

[000202] Other embodiments are set forth within the following claims