CA2431859C - Transgenic and cloned mammals - Google Patents

Abstract

The invention features methods of making cloned and transgenic mammals, e.g., goats. The methods include making a somatic cell line, e.g., a transgenic somatic cell line which can be used as a donor cell, methods of producing a cloned or transgenic mammal by introducing the genome of a somatic cell into an enucleated oocyte, preferably a naturally matured oocyte which is telophase, to form a reconstructed embryo, and methods of transferring the reconstructed embryo. The invention also includes cell lines, reconstructed embryos and cloned or transgenic mammals.

Description

50409-13D(S) TRANSGENIC AND CLOhIED-MAMMALS -- . Background of the Invention 'The ability to modify animal genomes through transgenic technology has opened new avenues for medical applications. By targeting the expression of biomedical .proteins to the mammary gland of large farm animals, low-cost production of high quantities of valuable therapeutic proteins is now possible.
Houdebine (i995) Reprod. Nutr. Dev. 35:609-617; Maga et al. (1995) -Bio/Technology, 13:1452-1457;.Echelard (1996) Curr.Op.Biotechnol. 7:536-540;
Young et_ al. ( 1997) BioPharm. 10:34-38. f~lthough the total sales for the top fifteen biopharmaceuticals in 1996 were $7.S~biIlion, expectations are that this number will continue to rise in the future. Med. Ad News 16:30.- Transgenic technology. is appl.icabie and attractive-for.proteins that, whether duewto.high unit,-dosage requirements, frequency of administration, or large patient populations, -are needed in high volume, and also to complex proteins that are difficult to produce in commercially viable quantities using traditional cell culture methods.
In addition, the production of human pharmaceuticals in the milk~of transgenic 20 farm animals solves many of the problems 'associated with microbial bioreactors, e.g., lack ofpost-translational modifications,.improper~folding, high purification costs, or animal cell bioreactors, e.g., high capital costs; expensive culture media, low yields. . . ~ - - -Dairy goats are ideal for transgenic production of therapeutic recombinant 25 proteins. Their average milk output is 600-800 liters per lactation. . With herds of WC 00126357 ~C°T.ItfS9912571~
a manageable size and at concentrations of approximately 1-5 gramslliter.
reproducibly achieved with various animal models, yields of transgenic protein to obtain 1-300 kg of purifaed product per year are achievable. Cordon et al.
(1987) BiolTechnology 5:1183-1187; l~eade et al. (1990) BiolTeclmology 8:443-446;
Ebert et aI. (I991) BiolTeclanology 9:835-838; Simons et aI. (1987) Nature 328:530-532; Vhright et al. {199.1 ) Bi~lTechnology 9:801-834; Velander et al:
. _.
(19-92) Proc Natl.4cact S'ci USA 89:12003-120007; Hansson et al. (1994) J73ioi Chem. 269:5358-5363; liurwitz et al. (1994) Transgenic lies. 3;365-375. 'This represents the low to middle range.of the high volume protein category and quantities that would be required for the majority of biopharrrr~aceuticals currently under development: Ivloreover, the goat generation interval, i.e., gestation, growth to sexual maturity and gestation, is 18 months as compared to almost three years for cows. This period permits expansion of the produetiora herds within the time frame needed for the regulatory approval of the transgenically-produced therapeutic proteins. rinaliy, the:rr~uch lower i:~cide:~ce of sc:apie i::
goats (only 7 cases ever reported in the tT.S.) relative to sheep, which have identical reproductive performance, and lower lactation output, makes goats better candidates for the production of therapeutic proteins. .
currently, there are very few reliable methods of producing transgenic goats. one sl~:ch :~etlrnd is prons~clea_r microinjection. Using pronuclear microinjection methods, transgene integration into the genetic snake up ~ccurs iri 1-3% of all the anicr~injected embryos. Ebert et al. (I993) Theriogenology, 39:121-135.
In 1981, it was reported that mouse embryonic stem cells can be isolated, propagated in viv~, genetically modified and, ultimately, can contribute to the germline of a host embry~. leans et al. (198i) Nature 292:154-156; ~lartirB
(1981) Proc Natl Acad ;Sci USA 78:?634-7638; Bradley et al. (1984) Nature 309:255-256. Since then, marine erruayonic stem cells have been extensively exploited in developmental and genetic steadies to modify, e.g., delete, replace, mutate, single targeted genes. I~Iansour et al. (1988) Nature 336:348-352;

50409-13D(S) McMahon et al. (1990) Cell 62:1073-1085; recently reviewed in: Bronson et al. (1994) J. Biol. Chem. 269:27155-27158;
Rossant, et al. (1995) Nat. Med. 6:592-594. Although extensive studies in the mouse have clearly indicated the utility of these elegant and powerful techniques, successful application of embryonic cell technology has been conclusively reported only in the mouse.
A need exists, however, for methods for obtaining cloned and transgenic animals such as goats.
Summary of the Invention The present invention is based, at least in part, on the discovery that cloned and transgenic mammals, e.g., cloned and transgenic goats, can be produced by introduction of a somatic cell chromosomal genome into a functionally enucleated oocyte with simultaneous activation. The functionally enucleated oocyte can be activated or nonactivated. In one embodiment, a nonactivated functionally enucleated oocyte (e.g., a caprine oocyte at metaphase II stage) is fused (e.g., by electrofusion) with a donor somatic cell (e.g., a caprine somatic cell) and simultaneously activated with fusion. In another embodiment, an activated functionally enucleated oocyte (e. g., a naturally matured caprine oocyte at telophase stage) is fused (e. g., by electrofusion) with a donor somatic cell (e.g., a caprine somatic cell) and simultaneously activated with fusion.
The use somatic cell lines, e.g., recombinant primary somatic cell lines, for nuclear transfer of transgenic nuclei dramatically increases the efficiency of production of transgenic animals, e.g., up to 1000, if the animals are made by the methods described herein. It also 50409-13D(S) solves the initial mosaicism problem as each cell in the developing embryo contains the transgene. In addition, using nuclear transfer from transgenic cell lines to generate transgenic animals, e.g., transgenic goats, permits an accelerated scale up of a specific transgenic line. For example, a herd can be scaled up in one breeding season.
Thus, in one aspect the present invention provides a method of producing a transgenic non-human mammal comprising: (a) introducing a nucleus from a non-human mammalian somatic cell into a functionally enucleated oocyte that has been pretreated with ethanol, said functionally enucleated oocyte being from the same species as said somatic cell and being in the metaphase II stage of meiotic cell division, and said nucleus from said somatic cell comprising at least one recombinant nucleic acid sequence under the control of at least one promoter sequence, to form a reconstructed embryo; and (b) allowing the reconstructed embryo from step (a) to develop into a mammal, thereby providing the transgenic non-human mammal.
In another aspect the present invention provides a method of making a transgenic non-human mammal comprising:
(a) fusing a non-human mammalian somatic cell capable of expressing a transgenic protein with a functionally enucleated oocyte that has been pretreated with ethanol, said functionally enucleated oocyte being from the same species as said somatic cell and being in the metaphase II
stage of meiotic division, and the nucleus from said somatic cell containing at least one recombinant nucleic acid sequence to obtain a reconstructed embryo; (b) activating the reconstructed embryo from step (a); (c) transferring the activated reconstructed embryo from step (b) into a female non-human mammalian recipient; and (d) allowing the - 3a -50409-13D(S) transferred reconstructed embryo from step (c) to develop into a mammal, thereby providing the transgenic non-human mammal .
In another aspect the present invention provides a method of making a transgenic non-human mammal comprising:
(a) fusing a non-human mammalian somatic cell capable of expressing a transgenic protein with a functionally enucleated oocyte, said functionally enucleated oocyte being from the same species as the somatic cell and being in the metaphase II stage of meiotic division, and the nucleus from said somatic cell containing at least one recombinant nucleic acid sequence to obtain a reconstructed embryo; (b) activating the reconstructed embryo from step (a); (c) maintaining the activated reconstructed embryo from step (b) in culture until the embryo is in the 2- to 8-cell stage of embryogenesis; (d) transferring the 2- to 8-cell stage reconstructed embryo from step (c) into a female non-human mammalian recipient; (e) allowing the transferred reconstructed embryo from step (d) to develop into a mammal thereby providing the transgenic non-human mammal.
In another aspect the present invention provides a method of producing a transgenic non-human mammal comprising: (a) introducing a nucleus from a non-human mammalian somatic cell into a functionally enucleated oocyte, said functionally enucleated oocyte being from the same species as said somatic cell and being in the metaphase II stage of meiotic cell division, and said nucleus from said somatic cell comprising at least one recombinant nucleic acid sequence under the control of at least one promoter sequence, to form a reconstructed embryo; (b) transferring said reconstructed embryo to a non-human mammalian recipient when said reconstructed embryo is in the - 3b -50409-13D(S) 2- to 8-cell stage of embryogenesis; and (c) allowing said reconstructed embryo to develop into a mammal, thereby providing the transgenic non-human mammal.
The generation of transgenic animals, e.g., transgenic goats, by nuclear transfer with somatic cells has the additional benefit of allowing genetic - 3c -WO 00/26357 1'~'T/TJS99125'710 manipulations that are not feasible with traditional rnicroinjcction approaches.
Foz example, nuclear transfer with somatic cells allows the introduction of specific mutations, or even the targeting of foreign genes directed to specific sites in the genome solving the problem of integration position effect. Homologous recombination in the dotaor s~r~aatic cells can °'knock-out" or replace the endogenous protein, e.g., a endogenous goat proteira, to lower purification costs _.
of heterologous proteins expressed in milk and help to precisely adjust the animal bioreactors.
In general, the invention features a method of providing a cloned non-human tiiammal, e.g:, a cloned goat. The methods below are described for goats, but can be applied for ~nli ror~-laurrnn mammal. The method includes:
introducing a ca~rine ger~orrac from a caprine.somatic cell into a caprine oocyte, preferably a naturally matured telopha~e oocyte, to form ~ reconstructed embryo;
t 5 an'd allowing the reconstructed embryo to develop into a goat, e.g:, by introducing the reconstructed embryo into a recipient doe, thereby providing a goat.
In one embodimeait, the nucleus of the caprine somatic cell is introduced into the caprine oocyte, e.g., by direct nuclear injectior$ or by fusion, e.g., electrofusion, of the soriiatic cell with the oocyte:
In preferred eynbodiment, the goat develops from the reconstructed embryo. In another embodiment, the goat is a descendant of a goat which developed from the reconstructed embryo.
In a preferred eubodiment: the somatic cell is non-quiescent {e.g., the cell is activated), e.g., the somatic cell is in G, stage. In another preferred embodiment, the somatic cell is quiescent (e.g., the cell is arrested),.e.g., the somatic cell is in G~ stage. In a preferred embodiment, the somatic cell is an embryonic somatic cell, e.g., the somatic cell is an embryonic fibroblast. The somatic cell can be any of: a fibroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumaxlus cell,.a neural cell or a mammary cell.

-4.

V1'O 00126357 PCTlUS99/25710 In a preferred embodiment; the oocyte is a frmction~lly enucleated oocyte, e.g., an enucleated oocyte. In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an. in vivo.protocol to obtain an oocyte which is in a desired stage of the cell cycle' e.g., metaphase II or telophase; the oocyte is activated prior to or simultaneously with the introduction ~f the genome. In another preferred emb~diment, the o~cyte and somatic cell are synchronized, e.g., both the oocyte and somatic cell are activated or both the oocyte and somatic cell are arrested:
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo with a second goat. A second goat can be a normal goat, a second goat which devel~ps from a reconstructed embryo or is descended.from a goat which developed from a reconstructed embryo or a second goat developed from a reconstructed embryo, or descended from a goat which developed from a reconstructed embryo, which was formed .
from genetic material from the same animal, ~n animal of the same genotype, or same cell line, which supplied the genetic material for the first goat. In a preferred embodiment, a first transgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which 2U tleveiOpeC1 fiom a. re~onStrllCteC1 emDryO anQ whlC:h LOI'liatri5 a different iaagtsg2ne that the first transgenic goat.
In a preferred embodiment, the goat is a male goat. In other preferred embodiments, the goat is a female goat. A female goat can be induced to lactate and milk can be obtained from the goat.
In a preferred embodimento a product, e.g., a protein, e.g:, a recombinant protein, e.g:; a human protein, is recovered from the goat; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, bI~od, skin or meat of the goat.
_5_ WO 00126357 PC'i'/TJS99125710 In another aspect, the invention features a method of providing a transgeriic non-human tnamrnal, e.g., a transgenic.goat. The methods below are described for goats, but can be applied for any non-human anamxnal. The method includes: introducing a genetically engineered caprine genome of a caprine somatic cell into,a caprine oocyte, preferably a naturally matured telophase oocyte, to form a reconstructed drnbryo; and allowing the reconstnacted embryo _e to develop into a goat, e.g., by introducing the xeconstructed embryo into a recipient doe; thereby providing ~ transgenic goat.
In one embodiment, the nucleus of the genetically engineered caprine somatic cell is introduced into the caprine oocyte, e.g., by direct,nuclear injection or by fusion, e.g., electrofusion, of the somatic cell with the oocyte.
In preferred embodiment, the goat develops from the se~onstn~cted embryo. In. another embodiment, the goat is a descendant of a g~at which developed from the reconstructed embryo.
In a preferred embodiment: the somatic cell is non-quiescent (e.g., the cell is activated), e..g., the somatic cell is in (~, stage. In another preferred embodiment, the somatic cell is quiescent (e.g.,-ihe cell is arrestEd), e.g., the somatic cell is in Go stage. In a preferred embodiment; the somatic cell is an embryonlC SOmatlC Cell, e.g., the somatic cell is an, embryonic fibroblast. A
2o somatic cell can be any of: a fbroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell or a mammary cell.
In a preferred embodiment, a transgenic sequence has been introduced into the somatic cell; the somatic cell is from a cell liras, e.g:, a primary cell line;
the somatic cell gs from a cell line and a transgenic sequence has been insexted into the cell.
In a preferred emlaodiment, the oocyte is a functionally enucleated oocyte, e.g., an enucleated ~ocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an in vivo pr~tocol to obtain an oocyte which is in a desired stage of the cell WO 00!26357 1~~:'1'~599125710 cycle, e.g., metaphase II or telophase; the oocyte is activated prior to or simultaneously with the.introduction of the genetically engineered genome. In another preferred embodiment, the oocyte and somatic cell are synchronized, e.g., both the oocyte and the somatic cell are activated or both the oocyte and somatic cell are arrested.
In a preferred embodiment, the method furtlher includes mating the _.
transgenic goat which develops from the reconstructed embryo with a second goat. The second goat can be a normal goat, a seccmd goat which develops from a reconstructed embryo or is descended from a goat which developed frorrfr a reconstructed embryo or a seeond goat developed from a reconstructed embryo, or descended from a goat which developed from a reconstructed embryo, which was formed from genetic material frorzr the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first goat.
Ima preferred embodiment, a first trarisgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which developed from a reconstructed embryo and which contains a different transgene than the first transgenic goat.
In a preferred embodiment, the goat is a m;~Ie goat. In other preferred embodiments the goat is a female goat. A female goat can be induced to lactate z0 and milk can be oniained from the goat.
In a preferred embodiment: a product, e.g.,. a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the goat; a product, e.g., a protein, e.g., a human protein, is recovered from tl~e milk, urine, hair, blood, skin or meat of the goat.
In a preferred embodiment, the caprine genome of the somatic sell includes a transgenic sequence. The transgenic sequence can be any of integrated into the genome; a heterologous transgene, e.g., a human transgene;
a knockout, knockin or other event which disrupts the expression of a caprine gene;
m a sequence which encodes a protean, e.g., a human protein; a heterologous 30. promoter; a heterologous sequence under the coni~ol of a promoter, e.g., a caprine _7_ WO 00126357 P°C't'IlQJS99/25710 promoter. The transgenic sequence care encode any product of interest such as a. .
protein, polypeptide or peptide. A protein can be any of: a hormone, an iminunoglobulin, a plas~iaa protein, and era enzyme. The transgenic sequence car!
encode any protein whose expression in the transgenic goat is desired including, but not limited to, any of a-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxade clisrnutase, fibrogen, glucocerebrosidase, ..
glutamate decarboxylase, humaru seaym albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythapoietin, tissue plasminogen activator, human growth factor; antithrombiri III, insulin, prolactin, and al-antitrypsiri.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a-preferred embodiment, the caprine genome includes a heterologous transgenic seguence.under the control of a promoter, e.g., a caprine promoter..
The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-speci~°ic prorrioters. The rrlilk-specific promoter can be any of. a casein promsiter9 a beta lactogl~bulin promoter, a whey acid protein promoter and a lactaUumin promote.
In another aspect, the invention features a method of making or producing a non-human mammal, e.g., a goat, e.g., a cloned ~r transgenic goat. The methods below are described for goats; but can ~e applied for any non-human matrimal.
The method includes fusing, e.g., by electrofusi~n, a caprine sorriatic cell, e.g., a caprine somatic cell capable of expressing a transgenic protein, with an erlucleated c~prine oocyte, preferably a naturally matured telophase oocyte, to obtain a reconstructed embryo; activating the reconstructed embryo;
transferring the embryo int~ a recipient doe; and allowing the embryo to develop into a goat.
_g_ w0 00/26357 1'~T~JS9gI25710 In a preferred embodiment, the goat develops from the reconstructed embryo. In another embodiment, the goat is a descendant of a goat which developed from the reconstructed embryo. .
In a preferred embodiment, the somatic cell is an embryonic somatic cell.
A somatic cell can be any of: a ~broblast (e.g., a primaary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell ox a mammary cell. In a ..
preferred embodiment, the somatic cell is a non-quiescent cell (e.g., the cell is activated), e:g., the somatic cell is ira G, stage, e.g., in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g., the somatic cell is in Go stage.
In a preferred embodiment, the oocyte is in metaphase II: Alternatively, the oocyte is in telophase. In either embodiment, the oocyte is activated prior to or simultaneously with the introduction of the genome. In a preferred embodiment; the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an in vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., metaphase II or telophase.. In a preferred embodiynent, the oocyte and somatic cell are synchronized, e.g., both the oocyte. and somatic cell are activated or both the oocyte and somatic cell are arrested.
In a preferred embodiment: a transgenic sequence has been introduced ~0 into the somatic ceii; the somatic ceii is from. a ceii line, e.g., a primary c;eil iir~d;
the somatic cell is from a cell line and a transgenic sequence has been inserted into the cell.
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo witlh a second goat. A second goat can be a normal goat, a second goat which develops from a xeconstructed embryo or is descended from a goat which developed from a reconstructed embryo or a second goat developed from a reconstructed embryo,. or is descended from a goat which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or 3Q . same cell Line, which supplied the genetic material for the first goat.
In a WO 00/263x7 1''C'f/U599/25710 preferred embodiment, a first transgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which developed from a reconstructed embryo and which contains a different transgene than the first transgenic goat.
In a preferred embodirhent, the goat is a male goat. In other preferred embodiments, the goat is a female g~at. I~ female goat can be induced to lactate and milk can be obtained frorri the goad.
In a preferred embodimerbt: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from.the goat; a product, e.g., a protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the goat.
In a preferred embodiment,: the caprine genome of the soraiatic cell includes a transgenic sequenee. The transgenic sequence can be any of integrated into the genome; a heterologous transgene, e.g., a human transgene;
a knockout; knockin or other event which disrupts the expression of a caprine gene;
a seyaence which Pncoc_les a protein, e.g., a human protein; a heterologous promoter; a heterologous sequenee under the control of a promoter, e.g.; a caprine promoter. The transgenic sequence can encode any product of interest such as a protein; a polypeptide, or a peptide. A protein can be any of: a hormone, an iinmunoglobulin, a plasma protein, and an enzyme.. i~he transgenic sequence can encode any protein whose expression in the transgenic goat is desired including, but not limited to any of a.-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen; glucocerebrosidase;
glutamate rlecarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator,,human growth factor, antithrombin III, insulin, prolactin, and al-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
_t~_ s WO 00/2b357 PCTltJS99J25710 In a preferred embodiment, the caprine genome comprises a heterologous transgenic sequence under the control of a promoter, e.g., a caprine promoter.
The promoter can be a tissue-specific promoter. The tissue specific promoter can be any o~ milk-specific promoters; blood-specific promoters; muscle-specific b . prorrioters; neural-specific promoters; stein-specific promoters; hair-specific promoters9 and urine-specific promoters. . The milk-specific promoter can be any ..
of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lacfalbumin promoter.
The invention also includes a non-human ani.nial made by any of the methods described herein. The methods described for goats can be applied for any non-human mammal. Accordingly, in another aspect, the invention features a cloned goat, or descendant thereof, obtained by introducing a caprine genome of a caprine'somatic cell into a caprine oocyte, preferably a naturally matured 75 telophase oocyte, to,a obtain reconstructed embryo and allowing the reconstructed embryo to develop into a goat.
In a preferred embodiment, the caprine genc~me can be frown an embry~nic somatic Belt. A somatic cell can be any of: fibroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell or a riiamrnary cell. In a preferred embodiment, the somatic cell is a non-quiescent cell (e.g. the cell is activated), e.g., the somatic cell is in G, stage, e.g., in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g:, the somatic cell is in Ga stage.
In a preferred embodiment, the caprine oocyte can be a functionally enucleated oocyte, e.g., an enucleated oocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an in vtvo protocol to obtain an oocyte which is in a desired stage of the cell .
cycle, e.g., metaphase II or telophase; the oocyte i s activated prior to or simultaneously with the introduction of the genome. In a preferred embodiment, -1 i-WO Q0/2b357 PC'flUS99i257i~
the oocyte and somatic cell,are synchronized, e.g., both the oocyte and somatic cell are activated or both the oocyte and somatic cell are arrested. _ .
In a preferred embodiment, the caprine genorne can be introduced by fusing; e.g., by electrofusion, of a somatic cell with the functionally enucleated oocyte.
In another aspect, the invention features one, or more, .e.g.; a population having at least one male and one female, cloned goat, each cell of which has its chromosomal genome derived from a caprine somatic cell, wherein said caprine somatic cell is from ~ goat other than cloned goat.
In a preferred embodiment, the chromosomal genome can be from an embryonic somatic cell. A somatic cell can be any of: a fibroblast (e.g., a primary- fibroblast), a muscle cell (e.g., a myocyte), a neural cell, a cumulus cell or a mammary celi. In a preferred embodiment, the somatic cell is a non-quiescent cell (e.g., the cell is activated), e.g., the Somatic .,211 is in ~, stage, ;,.g., in G, prior to ST1~IZT. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g., the somatic cell is iii Cso stage.
In another aspect, the invention features a transgenic goat, or descendant r_hP,,~of5 obtained by iz~txoducing a caprine genome of a genetically engineered caprine somatic cell into a caprine oocyte, preferably a naturally. matured telophase oocyte, to obtain a reconstructed embryo and. allowing the reconstructed embryo to develop int~ a goat.
In a preferred embodiment, the caprine genome can be from an embryonic soanatic cell. In another preferred emb~diment, the caprine genome can be froara a caprine fibroblast, e.g:, an embryonic fibroblast.
In a preferred embodiment, the caprine oocyte can be a functionally enucleated oocyte; e.g., an enucleated oocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in viyo protocol; the oocyte is obtained ~~2-WO 00/26357 PCTltIS9~/25710 using an ifi vivo protocol to obtain an oocyte which, is in a desired stage of the cell cycle, e.g., metaphase TI or telophase; the oocyte is activated prior to or simultaneously with the introduction of the genome. lai a preferred embodiment, the oocyte and the somatic cell are synchronized, e.g., both the oocyte and the somatic cell are activated or both the oocyte and the somatic cell are arrested.
In a preferred embodiment, the caprine genome can be introduced by fusing, e.g., by electrofusion, of a somatic cell with the functionally enucleated oocyte.
In a preferred embodiment, the caprine genome of the somatic cell includes a transgenic sequence. The transgenic sequence can be any of:
integrated into the genome; a heterologous transgenc~, e.g., a human transgene; a knockout, knockin or other event which,disrupts the expression of a caprine gene;
a sequence which encodes a protein, e.g., a human. protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode a protein which can be arty ~f a hormone, an immunoglobulin, a plasma protein, an enzyme, and a peptide. The transgenic sequence can encode any product of interest such as a protein, a polypeptide or a peptide. A protein which can be any protein whose expression in the transgenic goat is desired including, but not limited to any of a-I
, .proteinase inhibitor, aikaiine phosphotase, angiogenin, extraceiiulare s~iperoxide dismutase, fibrogen, glucocerebrasidase, glutamate decarboxylase, human serum albumin, myelin basic, protein; proinsulin, soluble (~~4, lactoferrin, lactoglobulin;
lysozyme, Iactoalbumin, erythrpoietin, tissue plasminogen activator, hmnan grovth.factor, antithrombin III, insulin, prolactin, and ocl-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a preferred embodiment; the caprine genome comprises a heterologous transgenic sequence under the control of a promoter, e.g., a caprine prombter.
The promoter can be a tissue-specific .promoter. The tissue specific promoter can be any of milk-specific promoters; blood-specific promoters; muscle-specific WO 00126357 PC"I'/U~991257t0 promoters; neural-specific promoters;, skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a Iactalbumin promoter. .
In another aspect, the invention features a transgenic goat, each cell of ..
which has its chromosomal genome derived from a genetically engineered caprine somatic ccll, wherein paid caprine somatic cell is from a goat other than said transgenic goat.
In a preferred embodiment, the chromosomal genome can be from an embryonic somatic cell. In another preferred embodiment, the chromosomal genome can be from a caprine .flbroblast, e.g., an embryonic fibroblast.
In a preferred embodiment, the chromosomal genome of the soriiatic cell includes a transgenic sequence. The transgenic sequence can be any of-.
integrated into the genorne; a heterologous transgene, e.g., a human transgene; a knockout; knockin or other event which disrupts the expression of a caprine gene;
a sequence vcihich encodes a protein,.e.g., a hur~nan protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode amy product of interest such as a 20 , protein, a polypeptide and a peptide. ~ ~roteiti can be aroy of: a hormone, an immunoglobulin, a plasma protein, an enzyme, and a peptide. The tiarisgenic sequence can encode any protein whose expression in the transgenic goat is desired including, but not limited to any of: a.-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, 25 glucocerebrosidase, glutamate decarbokylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, .
lactoalbumin, erytlirpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and ocl-antitrypsin.
In a preferred embodiment; the transgenic sequence encodes a human , 30 protein.
-t 4-!v0 00126357 ~e'~~599/25710 In a preferred embodiment, the chromosomal genorne comprises a heterologous transgenic sequence under the control of a promoter, e.g., a caprine promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of milk-specific promoters; blood-specific promoters;
muscle-specific promoters; neural-specific promoters; skin-specific promoters;
hair-specific promoters; and urine-specific promoters. The milk-specific --promoter can be any of a casein promoter, a beta laci:oglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect; the invention features a goat made by mating a goat which developed from a reconstructed embryo ~rnade; as described herein) with a second goat.
In a preferred embodiment: the second goat developed from a reconstructed embryo or is descended-fi-om a goat which developed from a reconstructed embryo; the second goat developed from a t°econstxucted embryo, or is descended from a goat which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell Line, which supplied the genetic material fox the first goat.
In a preferred embodiment, a first transgenic goat which develops from the Gu i~econ~t~c=ui~te~ eYfiW yCa ca~5'C~e ~~aicd ivitii a SeGvi'ad irnna"genii, goat which developed from a reconstructed embryo and which contains a different transgene than the first transgenic goat.
In another aspect, the invention features a plurality of fransgenic goats obtained by mating a goat which developed from a reconstructed embrym with a second goat.
In a preferred embodiment: the second goat developed from a reconstructed embryo or is descended from a goat v~rhich developed from a reconstructed embryo; the second goat developed from a reconstructed embryo, or is descended from a goat which developed from ~~ reconstructed embryo, which WO 00!26357 ~C'TAIJS99/257t~
was formed from genetic material fr~m the same animal, an animal of the same genotype, or same cell line, which supplied the genetic material for the first goat. , In a preferred embodiment, a first g~at which developed from a reconstructed embryo can be mated avith a second goat which developed from a reconstructed embryo and which contains a different transgene than the first goat:
In yet another aspect, the invention features a method of providing a transgenic goat which is hom~zygous for a transgenic sequence. The method includes providing a somatic veil which is heterozygous for a transgenic 10. sequence; alloying somatic.~ecombination to occur so as to produce a somatic cell which is homozygous for the transgenic sequence; introducing the genome from the somatic cell which is homozygous for the transgenic sequence into a caprine oocyte, preferably a naturally matured telophase oocyte, to fore a reconstructed embryo; and allowing the reconstructed embrya~.to develop into a 95 . goat, e.g:, by introduci~ag the reconstrzcted e~nhr~r:~ into a recipient doe, thereby providing a transgenic goat dvlcich is homozygous for a transgeriic sequence.
In another aspect, the invention features a transgenic goat which is homozygous for a transgenic sequence.
2o In a preferred embodiyraent, the transgenic goat was made by introducing the genome frown the sorttatic cell which is homozygous for the transgenic sequence into a caprine. oocyte, preferably a naturally matured telophase o~cyte, to form a reconstructed embryo; and allowing the reconstructed embryo fo develop into a goat.
In another aspect, the invention features a method of making a cloned non-human mammal, e.g., a goat, cow, pig, horse, sheep, Llama, camel. The method includes providing an activated oocyte, e.g., an oocyte in telophase stage, preferably a naturally matured telophase oocyte; fianctionaliy enucleating the . oocyte; introducia~g the chromosomal genome of a somatic cell into the CVO 00126357 t'C'6'1U599/25710 functionally enucleated oacyte to obtain a reconstructed embryo; and allowing the reconstructed embryo to develop ,e.g., by introducing the reconstructed embry~ into a recipient doe, thereby making a: cloned mammal.
In a preferred embodiment, the mammal, e.g., a goat, develops from the . recorsstructed embryo. In another embodiment, the mammal, e.g., a goat, is a descendant of a mammal, e.g., a goat which developed from the reconstrezcted ..
embryo:
In a preferred embodiment, the somatic cell is an embryonic somatic cell.
In a preferred embodiment the somatic cell is a fibrobiast, e.g., an embryonic fibroblast. In a preferred embodiment, the somatic cell is a non-quiescent cell (e.g., the cell is activated), e:g., the.somatic cell is in G, stage, e.g:, in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g:, the cell is arrested), e.g., the somatic cell is in Go stage.
In a preferred embodiment, the oocyte is. activated prior to or simultaneously with the introduction of the genorne. In a preferred emb~diment, the oocyte is obtained using an in vivo protocol, e.g., the oocyte is obtained using an in vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., metaphase Il or telophase. In a preferred embodiment, the oocyte and somatic cell are synchronized, e.g., both the oocyte ,and somatic cell are activated or both the oocyte and somatic cell are arrested.
In a preferred embodiment, the chromosomal genome of the somatic cell is introduced into the oocyte by fusion, e.g., electrofusion, or by direct injection of the nucleus into the oocyte, e.g., microinjection.
In another aspect, the invention features a cloned non-human mammal, e.g., a goat, cow, pig, horse, sheep, llama,' camel, obtained by functionally enucleating an activated oocyte, e.g., an oocyte in telophase, and introducing the chromosomal genome of a somatic cell into the enu.cleated oocyte, preferably a nat<irally matured telophase ~ocyte, to form a reconstructed embryo; and allowing 17_ WO 00/26357 P'C't'1US99l257t0 the reconstructed embryo to develop, e.g., by introducing the reconstructed embryo into a recipient mammal.
In a preferred embodiment, the oocyte is obtained using an in vivo protocol, e.g., the oocyte is obtained using an in viv~ protocol t~ obtain an oocyte:
which is in a desired stage of the cell cycle, e.g:,. telophase.
In another aspect, the invention features a reconstructed non-human mammalian embryo, e.g., a goat, cow, pig, horse, sheep, llama, camel embryo, obtained by functionally enucleating an activated oocyte, e.g., an oocyte in telophase, preferably a naturally ax~atured telophase oocyte, and introducing the chromosomal genome of a sorr~atic cell into the enucleated oocyte.
In a preferred embodiment, the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an irp vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., telophase In yet another aspect, the invention features a method of making a transgeriic non-human mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel.
The method includes providing an activated oocyte, e.g., an oocyte in telophase stage, preferably a naturally matured telophase oocyte; functionally enucleating 1 .-7_..,. s L,..-.~..,ni~, 7 ~ n~ o nsa ca4tn 11 7 Pt1 afi'~PPYPf~
Lo tile f5o(:ylG; ~it'Lr~uuc.ti3g cftc i,atavaaavsvaiaaa byenv~me va a gvnv...vt~... 3 ....g~.
somatic cell into the functionally enucleated oocyte to obtain a reconstructed eri~bryo; and allowing the reconstructed embryo to develop, e.g.; by introducing the reconstructed embryo into a recipient female, such that a transgenic mammal is obtained.
25 In a preferred embodira~ent, the maynmal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred embodiment, the somatic cell is an embryonic somatic cell.
In another preferred embodiment, the somatic cell is a fibroblast, e.g., an 3o embryonic fabrolalast. In a preferred embodiment, the somatic cell is a non-_~ g_ W~ OU126357 P~T/tJS991257I0 quiescent cell (e.g., the cell is activated), e.g., the somatic cell is in G, stage, e.g., in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g., the somatic cell is in. Go stage.
In a preferred embodiment, the oocyte is activated prior to or simultaneously with the introduction of the genuine. In a preferred embodiment, the oocyte is obtained using an io vivo protocol, e.g.; the oocyte is obtained-using an in vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., telophase. In a preferred embodiment, the; oocyte and somatic cell are synchronized, e.g., both the oocyte and somatic cell acre activated or both the oocyte and somatic cell are arrested. In a preferred embodiment, the chromosomal genome of the somatic cell is introducc;d into the oacyte by fusion, e.g., electrofusion, or by direct injection of the nucle~xs into .the oocyte, e.g., microinjection.
In a preferred embodiment, the nucleus ofthe somatic cell comprises a transgenic sequence. The. transgenic sequence can be any of: integrated intcs the genome; a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a caprme gene; a sequence e~Jhich encodes a protein, e.g.; a human protein; a heteroIogous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The 2o transgenic sequence can encode any product of inierest such as a protein, a polypeptide and a peptide. A protein can be any of a hormone, an immunoglobulin, a plasma protein, and an enzyme. The transgenic seqi.~ence can encode any protein whose expression in the transgenic mammal is desired including, but not limited to any of: a.-1 proteinase inhibitor, alkaline 25 phosphotase, angiogenin, extracellular superoxide dismutase; fabrogen;
giucocerebrosidase, giutarirnate decarboxylase, human serum albumin, myelin basic protein, proinsulin; soluble CDR, Iactoferrin, lactoglo~ulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and a.l-antitrypsin.
_1g_ WO OOI2b3S7 . YCT/U~a991257t0 In a preferred embodiment, the transgenic sequence encodes a human protein. _ In a preferred embodiment, the chromosomal genome comprises a heterologaus transgenic sequence under the control of a promoter, e.g., a mammalian-specific promoter, e.g.; a caprine promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of: mills-specific promoters; blood-specific promoters; muscle-specific promoters;
neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect, the invention features a transgenic non-human mammal, e.g:, a goat, cow, pig, horse, sheep; llama, cannel, made by functionally t5 erlucleating an activated oocyte, e.g., an oocyte.in telophase, preferably a naturally matured telophase ~ocyte, and introducing the chromosomal genome of a genetically engineered sorna~ic cell into the enucleated oocyte td form a reconstructed embryo and alloying the reconstructed embryo to develop, e.g., by introducing the reconstructed embryo int~ a recipient axyam~nal.
In a preferred embodiment, ft~e oocyte is obtained using an iiuvivo protocol, e.g., the oocyte is obtained using an iri vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., telophase.
In another aspect; the invention features a reconstructed non-human mammalian embryo, a:g., a goat, cow; pig, horse,,sheep, llama, camel embryo, obtained by functionally enucleating an activated oocyte, e.g., an oocyte in telophase, preferably a naturally matured telophase oocyte, and introducing the chromosomal genome of a genetically engineered somatic cell into the enucleated oocyte.

WO 00/2657 T'OT/tJS99/2571d In a preferred embodiment, the oocyte is obtained using an in vivo protocol, e.g., the oocyte is obtained using an ifr vivo protocol to obtain an odcyte which is in a desired stage of the cell cycle, e.g.; telophase.
In yet another aspect, the invention features a vrrrethod of making a cloned non-human mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The _.
method includes providing an oocyte, preferably a na~t~~rally matured telopliase oocyte; functionally enucleating the oocyte; introducing the chromosomal genome of a omatic cell into tlZe functionally enucleated oocyte to ~btain a reconstructed embryo, wherein the oocyte is activated prior to or simultaneously with the introductiorq ofthe chromosomal genome; introducing the reconstructed embryo into a recipient mammal; and allowing the reconstnacted embry~ to develop, thereby making a cloned mammal.
In a preferred embodiment, the mammal develops from fhe reconstructed 75 embryo. In another embodiment, the mammal is a descendant of a mamrilal ~~~hich developed from floe reconstructed embryo.
In a preferred embodiment, the somatic cell i;> an embryonic cell. In another preferred embodiment, the somatic cell is a fibroblast; e.g., an embryonic fibroblast. In a preferred embodiment, the somatic cell is a non-quiescent cell (e.g., the cell is activated), e.g., the somatic cell is in C.~, stage, e.g., iri G, prior to START. iri another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g., the somatic cell is iri (3o stage. In another preferred embodiment, the oocyte. is ain enucleated oocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in vivo prot~col, e.g., the.oocyte is obtained using an in vivo protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., telophase. In a preferred embodiment, the oocyte and somatic cell are synchronized, e.g.; both the oocyte and somatic cell are activated or both the oocyte and SOmatlG Cell are arrested.

WC? 00/2635'7 ~C'TIUS99I257t0 In a preferred embodiment, the chromosomal genome of the somatic cell is introduced into the oocyte by fusion, e.g., electrofusion, or by direct injection ofthe nucleus into the Qocyte, e.g., microinjection.
In another aspect, the invention features a cloned non-human mammal, e.gs~ a goat, cow, pig, horse, sheep, llama, carnal, made try functionally _.
enucleating a mammalian oocyte, preferably a naturally matured telophase oocyte, and activating the oocyte prior to or simultaneously ~.vith the introduction of the chromosomal genome of a somatic cell into the enucleated oocyte.
In yet another aspect, the invention features a. reconstructed non-human mammalian embryo, e.g., a goat, core, pig, horse, sheep, llama, camel embryo, obtained by functionally enucleating a mammalian oocyte; preferably a naturally matured telophase oocyte, and activating the oocyte prior to and/or simultaneously vrith tfe intrnduetis~l~ of the chromosol~la~ genolne of a somatic cell into the enueleated oocyte:
In another aspect, the invention features a method of making a transgenic non-human mammal, e.g., a goat, cow, pig, horse, sheep; llama, camel. The 2o method'includes providing an oocyte, preferably a naturally matured telophase oocyte; functionally enueleating the oocyte°, introducing the chromosomal genonie of a genetically engineered. somatic cell into the fiznctionally enucleated oocyte to obtain a reconstructed embryo, wherein the oocyte is activated prior to or simultaneously with the introduction of the chromosomal genome; and 25, allowing the reconstructed embryo to develop, e.g., by introducing the reconstructed embryo into a recipient mammal, such that a transgenic mammal is obtained.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal 30 ~ which developed from the reconstructed embryo.

w~ oons~s~ rcTr~s~gias~lo In a preferred embodiment, the somatic cell is an embryonic samatic cell.
In another preferred embodiment, the somatic cell is a fibroblast, e.g., an embryonic fibroblast. In a preferred embodiment, the somatic cell is a rion-quiescent cell (e.g., the cell is activated), e.g., the somatic cell is in G, stage, e.g., in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested, e.g., the somatic cell is in Go stage. In ..
another preferred embodiment, the oocyte is an enucleated oocyte In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is obtained using an in vivo protocol; the oocyte is obtained using an i~z viva protocol to obtain an oocyte which is in a desired stage of the cell cycle, e.g., metaphase Il or telophase; the oocyte is activated prior to or simultaneously with the introduction of the genome. In a preferred embodiment, the oocyte and somatic cell are synchronized, e.g., both the oocyte and the somatic cell are activated or both the oocyte and somatic cell are arrested.
In a preferred embodiment, the chromosomal genome of the somatic cell is introduced into the oocyte by fusion, e.g., electrofu.sion, ar by direct injection ofthe nucleus into the oocyte, e.g., rrlicroinjection.
In a preferred embodiment, the nucleus of the somatic cell comprises a trarisgenic sequence. The transgenic sequence can beg any of: integrated into the genome; a heteroiogous transgene, e.g., a human transgene; a knockaui, knuc;kin or other event which disrupts the_expression of a caprine gene; a sequence which .
encodes a protein, e.g., a human protein.; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a tissue-specific promoter.
The transgenic sequence can encode any product of interest such as a protein, a polypeptide and a pepride. A lirotein can be any of a hormone, an immunoglobulin, a plasma protein, and an enzyme. The transgenic sequence can encode a protein whose expression in the transgenic mammal is desired including, but not limited to any of: oe-1 proteinase inhibitor, alkaline -.
phosphotase, angiogenin, extraceliular superoxide dismutase, fibrogen, 30: glucocerebrosidase, glutamate decarboxylase, human senzm albumin, myelin WO 00!26357 ~'~T/1I~99/25710 basic pratein, proinsulin, solaable ~D4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogen activator, Human growth factor, antithrombin III, insulin, prolactin, and a 1-antitrypsin.
In a preferred embodiment, the transgenic sequence enc~des a human -protein.
In a preferred embodiment; the chromosomal genome comprises a heterologous transgenic sequence under the control of a promoter, e.g:, a mammalian-specific promoter; e.g., ~. caprine promoter: °The promoter can be a tissue-specific promoter. q'he tissue specific promoter can be any-of: triilk-specific.promoters; blood-specific promoters; muscle-specific .promoters;
neural-specific promoters; skin-specifie pr~moters; hair-specific pramoters;.and urine-specific promoters. The milk-specific promoter can be any of. a casein promoter, a beta lactoglob~alin promoter, ~ whey acid protein promoter and a lactalbumin promoter.
In another aspect, the inventiort features a transgenic non-human mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel; made by functi~nally enucleating a mammalian oocyte, preferably a naturally matured tc;lophase oocyte, and activating the oocyte prior to or simultaneously with the introduction of the chromosomal genc~me of a genetically engineered somatic cell into the enucleated oocyte.
In yet another aspect, the invention features a reconstructed non-human mammalian embr<,~a, e.g:, a goat, cow, pig, horse, sheep, llama, camel err~bryo, obtained by functionally enucleating a mammalian oocyte,.preferably a naturally matured telcrphase oocyte, and activating the oocyte.prior to or simultaneously with the introductioxi of the chromosomal genome of a genetically engineered somatic cell into the enucleated oocyte.
-vv~ oa~263s~ ~~~-r~~~~ns7~o The invention also includes a product, e.g., a protein, e.g:, a heterologous protein, described herein obtained from a nan-human mammal, e:g., a cloned or trarisgenic rriarnrnal, e.g., a cloned or transgenic goat, described herein.
In a preferred embodiment, product is milk or a pratein secreted into milk.
In another aspect, the invention features a method of providing a protein, e.g., a human protein. The method includes: providing anon-human mammal, e.g., a transgenia mammal, e.g., a transgenic goat; described herein; and recovering the product from the mammal, or from a product, e.g., milk; of the mammal.
In another aspect, the in~~en~ion features a me~tr~od of providing a heterologous polypeptide. The methods-includes introducing a caprine genome, e.g., by introducing a nucleus; of a genetically engineered caprine somatic cell into a caprine oocyte, preferably a naturally matured telophase oocyte, to form a reconstructed embryo; allowing the reconstructed eit~ibryo to develop into a goat, e.g., by introducing the reconstructed erhbryo into a recipient doe; and recovering .
the polypeptide Pram the goat or a descendant thereof.
In a preferred embodiment, the nucleus of the: caprine somatic cell is introduced into the caprine oocyte, e.g., by direct nuclear injection ~r by fusion, 2t? . e.g., electrofusion, of the somatic cell with the oocyte.
In a preferred embodiment: the somatic cell is anon-quiescent cell (e.g., the cell is activated), e.g., the somatic cell is in G, stage, e.g., in G, prior to START. In another preferred embodiment, the somatic cell is a quiescent cell (e.g., the cell is arrested), e.g:, the somatic cell is in d:~o stage. In a prefera-ed embodiment, the samatic cell is an embryonic somatic cell, e.g., an embryonic fibroblast. The somatic cell can be a fibroblast (e.g.., a primary fibroblast), a muscle cell (e.g., a myocyte), a neural cell, a cumulus cell or a mammary cell.
In a preferred embodiment, a transgenic sequence has been introduced into the somatic cell; the somatic cell is from a cell line, e.g., a primary cell line;

WO 00!26357 t'CTltJS9~lz~7i6D
the somatic cell is from a cell line and a transgenic sequence has been inserted into the cell.
In a preferred embadinmn~, the oocyte is a functionally enucleated oacyte, e.g;, an enucleated oocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in telophase; the oocyte is abtained using an iaa vivo protocol; the oocyte is obtained .~
using an in vevo protocol to obtain an oocyte which is in a desired stage of the cell cycle, a:g., metaphase II or talophase; the oocyte is activated prior to or.
simultaneously with the introductioa~ of the genome. In a preferred embodiment, the oocyte and the somatic cell are synchronized, e.g., both the oocyte and the somatic cell are activated or both the ooeyte and the somatic cell are arrested.
In a preferred embodiment, the caprine genome of the somatic cell includes a transgenic sequence. The transgenic sequence can be any of:
integrated into the genome; a heterologous transgene, e.g., a human transgene;
a .
knockout, knoclcin or o'iner event which disrupts the expression of a caprine gene;
a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest including a protein, a polypeptide and a peptide, A protein can be any of: a hormone, an im???ay_n_oglol?olin, a plassrxa pxotein, aid axc_ enayrue, Tb~e tsansgex~ic sequence cart encode any protein whose expression in the txansgenic goat is desired including, but not limited to any of a.-I proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular supcroxide dismutase, frbrogen, glucocerebrosidase, glutamate decarboxylase, human senzm albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythxpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, pralactin,,arid cxl-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
-2~-'WfJ 00/2635? PC'1"/1J~99/25710 lri a preferred embodinxent, the caprine genoinc; comprises a heterologous transgenic sequence under the control of a promoter, e.g., a caprine promoter.
The promoter can be a tissue-specific promoter. The tissue specific promoter can be . any of: milk-specific promoters; blood-specific promoters; muscle-specific promoters; neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be any ..
of: a casein promoter, a beta Iactoglobulin promoter, a whey acid protein promoter and a lactaibumin prompter.
In another aspect, the invention features a method of making a heterologous polypeptide. The method includes fusi;rag a genetically engineered caprine somatic cell which comprises a transgene encoding a heterologous polypeptide and a milk-specific promoter, with an enucleated caprine oocyte, preferably a naturally matured telophase oocyteg to obtain a reconstructed 15 embryo; and allowing the reconstructed erribryo to develop into .a ~rar~sgcnic goat, P.g., by introducing the reconstructed embryo into a :recipient doe:
In a preferred embodiment, the transgene is operatively linked to the milk-specific promoter. The milk-specific promoter can ~~e any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a, lactalbumin 20 promoter. .
~n a preferred embodiment, the nucleus of the caprine somatic cell is introduced into the caprine oocyte, e.g., by direct nuclear injection or by fusion, e.g., electrofusion, of the somatic cell with the oocyt e.
In a preferred embodiment, the somatic cell ::is an embryonic somatic cell.
25 In another preferred embodiment, the somatic cell is a fibroblast; e.g., an embryonic fibroblast.-Tn a preferred embodiment, the oocyte is a functionally enucleated oocyte, e.g., an enucleated oocyte.
In a preferred embodiment, the oocyte is in metaphase II; the oocyte is in ~~ telophase; the oocyte is obtained using an in vivo protocol; the oocyte is obtained WO ~0126357 P~~'/U5991257i8 using an in vivo protocol to obtain an, oocyte which is in a desired stage of the cell cycle, e.g., metaphase II or telophase; the oocyte is activated prior to or simultaneously with the introduction of the genorr~.e. In a preferred embodiment, the oocyte and the sornatie cell are synchronized, e.g., both the oocyte and the somatic cell are activated or both the oocyte and the somatic cell are arrested. .
In a preferred errabodiment, the caprine genome of the somatic cell --includes a transgenic sequence. The transgenic sequence can be any of:
integrated into the ger~ome; a heterologous transgene, e.g,, a human transgene; a knockout, knockin or other even'c wvhich disrupts the expression of a caprine gene;
a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest such as a protein, a polypeptide or a peptide. l~ protein can be any of a hormone, an immunoglobulin, a plasma protein, an erizyme. The trar~sgenic sequence can encode a protein whose expression in the-transgenic goat is desired including, 'out not limited'to any of: a-1 p~oteinase inhibitor, alkaline phosphcitase, angiogenia~, extraceliular superoxide dismutase, fabrogen, glucocerebrosidase, glutamate decarboxylase, human serum ,albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactogiobulin, lysozyme, lactoalbumin, erythrpoietin, tissue 7~1 pia_cmi_n_ngg~i a~tzva_tngi ~~~~~,n growth fa~tn_r~ antitl~gr~_rn__hi_n IIh i~cpl_ir_~3 ~_rnl_~r_.fia_t, .
and oc1-arttitrypsin.
In a preferred embodiment, the txansgenic sequence encodes a human protein.
In a preferred embodiment, the heterologous polypeptide is purified from ~ the milk of the transgenic goat.
In a preferred embodiment, the method can also include rrr~ilking the trans.genic goat.
In another aspect, the irwention features a method of providing a . heterologous polypeptide. Tl~e method includes obtaining a goat made by wo aorz~~s°r ~~Tr~s~~~2s~~o introducing a caprine genome of a genetically engineered caprine somatic cell . into a caprine oocyte, preferably a naturally matured telophase oocyte, to form a reconstructed embryo; and alloying the: reconstructed embryo to develop into a goat, e.g., by introducing the reconstructed embryo into a recipient d~e; and recovering the polypeptide from the goat, e.g., from the milk of the goat, or a descendant thereof. _.
In a preferred embodiment, the caprine geno:rrze of the somatic cell includes a transgenic sequence. Thc; transgenic. sequence can be any of:
integrated into the genome; a heterologous transgene, e.g., a human transgene;
a knockout, knockin or other event which disrupts the expression of a caprine gene;
a sequence which encodes a protein, e.g., a human protein; a l~eterologous promoter; a. heterologous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest such as a protein, a polypeptide and a peptide. A protein can be any of a horanone, an immunoglobulin, a plasma protein, and an enzyme: The transgenic sequence can encode any protein whose expression in the transgenic goat is desired including, but not limited to any of a.-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen; glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, 2o soluble t;u4, iactoferzrin, lactogiobuiin, iysozyrne, lactoaibumin, etytrirpoieiiir, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and cxl-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a 'human protein.
In a preferred ernbodirnent, the heterologous polypeptide is purred from the milk of the transgenic goat.
In another aspect, the invention features method ofmaking a reconstructed caprine embryo. The method includes introducing a caprine dvc~ oorz6~s~ ~e~r~s~~rzs~~o genorne from a caprine somatic cell into a caprine oocyte, preferably a naturally matured telophase oocyte, thereby forming a reconstructed embryo.
In a preferred embodiment: the somatic cell is a non-quiescent cell (e:g., the cell is activated), e.g.; the somatic Bell is in G, stage. .In, anothex preferred embodiment; the somatic cell is a. quiescent cell (e.g., the cell is arrested), e.g., the somatic cell is in Ga stage. In a preferred embodiment, the somatic cell is an -embryonic somatic cell, e.g., are embryonic fibrol~last. The somatic cell can be a fibroblast (e.g:, a primary fibroblast~, a muscle call (e.g., a myocyte), a neural cell, a cumulus cell .ar a mammary dell.
In a preferred embodiment: the ~ocyte is in_ metaphase II; the oocyte is in telophase; the oc~cy~e is obtained easing an in viv~ pros~col; the occyte is obtained using an in viva protocol to obtai~a an oocyte which is in a desired stage of the cell cycle, e.g., metaphase II of telophase; the oocyte is enucleated. In a preferred erribodiment; the oocyte and the soanatic cell are synchronized, e.g., both the oocyte and the somatic cell aro activated or both the oocyte and the somatic cell are arrested.
In yet another aspect; the invention features a reconstructed capriiae embryo obtained by inCroducing a capririe genome from a caprine somatic cell 2o into a caprine oocyte, preferably a naturally matured telophase oocyte.
In another aapect,, the invention features a method ofnnaking a reconstructed trans~enic caprine embryo. 'fhe anethod includes introducing a caprine genome, e.g_, by introducing a nucleus, of a genetically engineered 2~ caprine somatic cell into a eaprine ~~cyte, preferably a naturally matured telophase oocyte, thereby forming a transgenic reconstructed embrya.
In a preferred ernbodirnent the somatic cell is a non-quiescent cell ~i.e., the cell is activated), e.g., the somatic cell. is in G; stage. in another preferred embodiment, the sorna.tic cell is a quiescent cell ~a.e., the cell is arrested), e.g., the 30 somatic cell is in GQ stage. In a preferred embodiment, the.samatic cell is an ~30-WO 04/26357 t'CTigJS99I2571U
embryonic somatic cell, e.g.,. an embryonic fibroblast: The somatic cell can be a fibroblast {e:g., a prirriary fibroblast), a muscle cell (e.g., a rnyocyte>, a neural cell, a cumulus cell or a mammary cell.
In a preferred embodiment: the oocyte is in metaphase II; the oocyte is in . telophase; the oocyte is obtained using an in vivo protocol;-the oocyte is obtained using an are vivo protocol to obtain an omcyte which is in a desired stage of the cell --cycle, e:g., metaphase II or telophase; the oocyte is enuclea~ed. In a preferred embodiment, the ooeyte and the somatic cell are syrmhronized, e.g:, both the oocyte and fhe somatic cell are activated or both the oocyte and somatic cell are arrested.
In another aspect; the invention features a reconstructed transgenic caprine embryo obtained by introducing a caprine genome, e.g., by introducing a nucleus, of a genetically engineered caprine somatic cell into a caprine oocyte, preferably a naturally matured telophase oocyte.
Iri another aspect, the invention features a method of providing a herd of goats. The method includes making a first goat by :introducing a caprine genome;
e.g., by introducing the nucleus, from a caprine omatic cell into a caprine oocyte, ~,r, o~ r"i.,t. +" tt f, a +..t.. t.: t ,.. r ~,-...<aod ,~~."t,..«o Gv prv.tama4.a~l a'nasuramy Waa~tlr~"u a~:avptaaS2 vvvy e, w mi~"ii a reCOiass.aw.m o.asavay and allowing the reconstructed embryo to develop into the first goat; making a second goat by introduciing a caprine genorne, ~e.g., by introducing the nucleus, from a caprine somatic cell into a caprine oocyte, preferably a naturally matured, telophase oocyte, to form a reconstructed embryo and allowing the reconsiructed embryo to develop into the second gout; whereby the genome of the first and second goats aie from the genetic material of the same anneal, same genotype or same cell line, thereby providing a herd of goats.
In a preferred embodiment, the first goat, or' descendant thereof, is mated with the second goat or a descendant thereof.
_31_ WO OOI26357 PCT//tJS99125?nc~
In another aspect, the invention features a herd of gaats,obtained by making a first goat by introducing a capririe genome, e.g., by introducing the nucleus, from a caprine somatic cell into a caprine oocyte, preferably a naturally matured teloplzase oocyte, to form a reconstructed ernt~ryo and.allowing the reconstructed embryo to develop into the first goat; making a second goat by introducing a caprirde genome, ~.g.; by introd~acirig the nucleus; from a.capririe somatic cell into a caprine oocyte to form a reconstructed embryo and allovsring the reconstructed e~nb~yo to develop into the second goat; whereby the genorue of the first and second goats are from the genetic material of the same animal, same 90 genotype or same cell line.
In a preferred embodiment, the herd of goats is obtained by any of the methods described herein.
In another aspect, the invention features, an embryonic or fetal caprine somatic cell.
Ire a preferred em~bodin~aea~t, the cell is a purified embryonic or fetal cagrine somatic cell.
In a preferred embodiment, the cell is in a preparation of embryonic, or fetal caprine somatic cells.
In a-preferred embodiment, -the cell can be used to derive an embryonic or fetal caprine somatic cei't line.
Xn a preferred eynbodiment, the cell includes a transgene, e.g., a transgene encoding a polypeptide. 'hhe transgene can be: integrated into the genome of the satnatic cell; a heterologous transgene,.e.g., a heterologous transgene which includes a human sequence; a knockout, knoekin or other event which disnapts the expression of a caprine gene; a sequence which encodes .a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter: The transgenic sequence can encode a product of intexest such as a protein, polypeptide ar peptide.

CVO flfl/26~57 PC'fItJS99/2571fl W a preferred embodiment; the transgene encodes any of. a hormone, an imrnunoglobulin, a plasma protein, and an enzyme: 'the transgene can encode, - e.g:, any of a-1 proteinase inhibitor, alkaline phospl~~ofase; angiogenin, extracelluiar superoxide dismutase, fibrogen, glucocc:rebrosidase, glutamate decarboxylase, human serum albumin; myelin basic :protein, prainsulin, soluble CT34; lactoferrin, lactoglobulin, lysozyrrie, lactoalliutnin,.erythrpoietih, tissue plasminogen activator, human growth factor, antithromlain III, insulin;
prolactin, and al-antitrypsin.
In a preferred embodiment, the transgene is under the control of a 9 0 promoter, e.g., a heterologous or a caprine promoter.. The pramoter' can be a:
tissuerspecific promoter. The tissue specific promoter can be ariy of a milk-specific promoter; a blood-specific promoter; a muscle-specific promoter; a neural-specific promoter; a skin-sp~cifc promoter; a hair specific promoter;
and, a urine-specific promoter. The a ilk-specific promoter cai~ be, e.g., any of:
a Vii-casein promoter; a ~-Iactoglobin promoter; a whey acid protein ~iornoter; aid a lactalbumin promoter.
In' a preferred embodiment, the somatic cell :°~s a fibroblast. 'I'he fibroblast can be a primary fibroblasf or a primary derived fibrobiasf.
In a preferred embodiment, the cell is obtained from a goat; e.g., an embryonic goat, derived from a germ cell.obtained 4i'om a transgenic mammal.
The germ cell can be sperm from a transgenic goat.
In a preferred embodirr~ent, the cell is a genetically engineered embryonic or fetal caprine somatic cell, e.g., a purified genetically engineered embryonic or fetal caprine somatic cell.
In a preferred embodiment, the cell is.part of a preparation of genetically engineered embryonic or fetal caprine somatic cells. In another preferred embodiment, the cell is used to derive a genetically engineered embryonic or fetal caprine somatic cell Line.
In a preferred embodiment, the genetically f;ngineered cell includes a nucleic acid, e.g., a nucleic acid encoding a polype~atide, which has been wo oorz63s~ pC~ius~~lzs~g~
introduced, into the cell. T'he nucleic acid can be: integrated into the genome of the somatic cell; a heterologous rfucleic acid, e.g., a heterologous nucleic acid , which includes a human sequence; a knockout, knockin or other event which disrupts the expression of a caprine gene; a sequence which encodes a protein, ~ e.g., a human protein; a hetezologous promoter; a heferologous sequence under the control of a promoter, e.g., a caprine promoter.- The .nucleic acid sequence. can _.
encode any product of interest such as a protein, polypeptide or peptide.
Tn a preferred embodiment, the nucleic acid encodes atiy of a hormone, an immunoglobuliri, a plasma protein, and an enzyme. -The nucleic acid can 10~ : encode, e.g., any of cx-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucoceiebrosidase, glutamate decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysazyme, lactoalbumin, erythrpoietin, tissue plasminogen activator; human growth. factor, antithrombin iIl, insulin, prolactin, arid oil=antitrypsin.
In a preferred embodiment, the nucleic acid is under the control of a.
promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of a milk-specific promoter; a blood-specific promoter; a rriuscle-specific promoter; a neural-2t~ specific promoter; a skin-specific promoter; a hair specific promoter;
and, a urine-specific promoter. The milk-specific promoter can be,,e.g_, any of a (3-.casein promoter; a ~i-lactoglobin promoter; a whey acid protein promoter; and a lactalbumin promoter.
In a preferred embodiment, the somatic cell is a, fi~roblast. The fibroblast can be a primary fibroblast or a primary derived fibroblast.
In a preferred embodiment, the cell is obtained from a goat, e.g., an embryonic goat, derived from a germ cell obtained from a txansgenic goat. The germ cell can be sperm or an ooeyte from.a transgenic goat.
Tri a preferred embodiment, the cell is used as a source of genetic material far nuclear transfer -vvo oon6~s~ ~cTlus~~ns~lo In another aspect, the invention features an embryonic or fetal caprine ' sar~atic cell, a preparation of cells, or an embryonic or fetal caprine somatic cell line, e.g.; as described herein,, in a container, e.g., an airtight or liquid tight container.
In another aspect, the invention features an e~,mbryonic or fetal caprine somatic. cell, a preparation of cells, or an embryonic ar fetal caprine somatic cell line, e.g,, as described herein, which is. frozen, e.g., is cryopreserv~ed.
In another.aspect, the invention features a kit. The kit includes a container of the cell or, cells described.herein. In a preferred embodiment, the kit further includes,instructions for use in preparing a transgenic animal In a preferred embodiment; the kit furtherincludes a recipient oocyte, e.g., an enucleated oocyte.
In another aspect, the invention features a method for providing a component for the production of a cloned or transgenic goat. The method includes obtaining a frozen sample of the cell or cells, e.g., those described herein, and thawing the sample.
In another aspect, the invention features, a method of .preparing an embryonic or fetal caprine somatic cell line.. The method includes obtaining a somatic cell.from.an embryonic or fetal goat; and; culturing the cell, e,g., in a suitable medium, such that a somatic cell line is obtained.
In a preferred embodiment, the cell line is a. genetically engineered cell line,. e.g., the cell comprises a transgene. The transgene can be; integrated into the somatic cell genome; a heteroiogous transgene, e.g., a heferologous transgene which includes a human sequence; a knockout, knockin or other event which disrupts the expression of a caprine gene; a sequence which encodes a protein, 30. e.g., a human protein; a heterologotis promoter; a heterologous sequence under W~ 00126357 ~ t'~'t'~~99/257~0 the control of a promoter, e.g.,, a caprine promoter. The transgenic sequence can -encode ariy product of interest such as a protein, poiypeptide or peptide. .
In a preferred em'oodiment, the transgerie encodes any of: a hormone, an imtnunoglobulin, a plasma protein, and an enzyrrie. The transgene can encode any protein; e:g:, any of: 0:-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, ~brogen, glucocerebrosidase, ..
glutamate decarboxylase; human serum albumin, 'myelin basic p~oteii~, proinsulin, soluble CD4, lactoferrin, lacfbglobulin; lysozyme, lactoalbumin, erythrpoietin, tissue plaszninogen acti~~tor, human growth factor, antithrornbin III, insulin, prolactin, and oc 1-antitrypsin.
In a pref~~red ernbodiriient, the transgene is under the control off. a promoter, e.g., a caprine or.hetei~ologous promoter: The promoter can be a tissue specific promoter. °fhe tissue specific promoter can be any of a milk-specific promoter; a blood-specifac pi~~moter, a muscle-specific promoter; a neural-~5 specific p rorraoter; a skir~~speci~e pr~m,oter; a lair specify prompter;
and, a urine«
specific prornot~r. The milk-specific 'promoter can be, e.g., any of a ~3-casein promoter; a ~3-lactoglobin prorrfoter; a rwhey acid pratein promoter; and a lactalbuinin promoter.
In a preferred eanbodiment, the genetically engineered cell includes a nucleic acid, e.g., a nucleic acid encoding a polypeptide, which his been introduced into the cell. Tlie nucleic acid care be: integrated into the genome of the somatic cell; a heterologous nucleic acid, e.g:, a heterologous nucleic acid which includes a human sequence; a knockout, knockin or other event which disrupts the e~cpression of a caprine gene; a sequence which encodes a protein, e.g:, a human protein;.a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine promoter. The nucleic acid sequence can encode any product of interest such as a protein, polypeptide or peptide.
In a preferred embodiment, the nucleic acid can encode any of a hormone, an immunoglobulin, a plasma protein, and an enzyme. The nucleic acid can encode, e.g.; any of: c~-1 proteinase inhibitor; alkaline phosphotase, Wfl OOIZb357 PCTltJS99I25710 angiogeniti, extracellular superoxide dismutase, flbrogen; glucocerebrosida~e, glutamate decaxboxylase, human serum albumin, myeiiri basic protein, pxoinsulin, soluble CD4, Iactoferrin, lactoglobulin, Iysozyme, lactoalbumln, erythrpoietin, tissue plasminogen activatoz, human growth factor, antithrornbin III, insulin;
prolactin, and ixl-antitrypsin.
in a preferred embodiment, the rfucleic.acid is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be .a tissue-specific promoter; The tissue specific promoter can be any of a milk-specie promoter; a blood-specific promoter; a muscle-specific promoter; a neural-specific promoter; a skin-specific promoter; a hair specific promoter; and, a urine-specifie promoter. The rriilk-specific promoter can be, e.g., any of a [I-casein promoter; a ~3-lactoglobin promoter; a whey acid protein promoter; and a lactalbumin promoter.
In a preferred embodiment, the sarnatic,cell is a Iibroblast. The.fibrobiast can ~i 5 be a primary fibroblast or a primary derived fibroblast.
In a preferred emhodiment, the cell is obtained from a gaat, e.g., an embryonic or fetal goat, derived from a germ cell obtained from a transgenic goat. The germ cell can be sperm or an oocyte fronn a transgenic goat.
In a preferred embodiment, the cell is used as a source of genetic material . for nuclear transfer.
In another aspect, the invention features, a method of preparing an embryonic or fetal caprine somatic cell line. The method includes inseminating a female recipient with the semen fromv a goat; obtaining a transgenic, embryo from the recipient; obtaining a somatic cell from the embryo; and, culturing the cell in a suitable medium, such that a somatic cell line is obtained.
In a preferred embodiment, the semen is from a transgenic goat.
In a preferred embodiment, the cell line is a genetically engineered cell line, e.g., the cell comprises 'a trans,gene. The transgene can be: integrated into the somatic cell genome; a heterologaus transgene, e.g., a heterologous transgene WO OOt26357 PC'T/~.7~9~/~~7t0 which includes a human sequence; a knockout, knoekin or other event which disrupts the expression of a caprine gene; a sequence which encodes a protein, e.g., a human protein; a heterotogous -promoter; a heteroiogous sequence under the control of a promoter, e.g., a caprine promoter. The transgenic sequence can encode any product of interest such as a pxotein, polypepfide or peptide. .
In a preferred embodiment, tlae transgezie encodes any of: a horrraone9 an ..
immunoglobulin, a plasrria protein,. and an enzyme. The transgene can encode any protein, e:g., any of oc-l; pxoteinase inhibitor, alkaline phosph4tase, -angiogenin, extraceilular superoxide dismutase, flbrogen, glucocerebrosidase, 9.0 glutamate decarboxylase, human serum albumin, myeliwbasic protein, proinsuliri, soluble CD4, lactoferrin,.lactoglobulin, lysozyme, lactoalbumin, erythrpoietin, tissue plasminogeti activator, hurnari growth factor, antithroznbin III;
insulin, prolactin, and a.l-antitzypsin.
In a preferred embodiment, the transgene is under the control of a Z~ promoter, e:g., a capr~ne os heterologous promoter. The_ promoter can be a tissue-specific promoter.. The tissue specific pxornoter can be any of: a milk-specific promoter; a blood-specific promoter; a muscle-specific promoter; a neural-specific promoter; a skin-specific promoter; a hair specific promoter; arid, a urine-specific pxomoter: The milk-specific promoter can be, e.g:, any of: a ~-casein ~0 promoter; a 6..lactoglobin promoter; a whey acid protein pr~moter; and a lactalbumin promoter.
In a preferred embodiment, the genetically engineered cell includes a nucleic acid, e.g., a nucleic acid encoding a palypeptide, which has been introduced into the cell. °The nucleic acid can be: integrated into the genome of 25 the-somatic cell; a heterologous nucleic acid, e:g., a heterologous nucleic acid which includes a human sequence; a knockout, knoclcin or other event which disrupts the expression of a eaprine gene; a sequence, which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e. g., a caprine promoter. . The nucleic acid sequence can a 3o encode any product of interest such as -a protein, polypeptide or peptide..
-38~

WO 00126357 l'CTffJS99/25710 In a preferred embodiment, the nucleic acid can encodes any of: a hormone; an immunoglobulin, a plasma protein, and an enzyme: Tlie nucleic . acid can encode, e.g., any of a.-1 proteinase inhibitor, alkaline phosphotase, _ angiogeniri,.extracellular superoxide dismutase, fibrogen, glucocerebrosidase;
glutamate decarbaxylase, human serum albumin, myelin basic protein, proinsulin;
soluble ~CD4, Iactoferrin, lactoglobulin, Iysozyme, lactc~lburi~in, erythrpaietin, ..
tissue. plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and al-antitrypsin.
In a preferred embodiment, the nucleic acid is under the control at' a ~o promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any af: a milk-specific promoter; a blood-specific promoter; a muscle-specific promoter; a neural-specific promoter; a skin-specific promoter; a hair specific piomoter; and, a urine-specific promoter. The milk-specific promotef can be, e.g., any o~ a ~3-casein promoter; a (3-lactoglobin promoter; a.whey acid protein promoter; and a lactalburilin promater_ . . , In a preferred embodiment, the somatic cell is a fibroblast. , The fibroblast can be a primary fibroblast or a primarjr derived fibroblast. .
In a preferred embodiment, the cell is used as a source of genetic material z0 for nuclear transfer.
The present invention is also based, in part, on the discovery that a reconstructed embryo which is transferred into a recipient mamrxial at th.e two to four cell stage of embryageriesis can develop into a cloned rnaminal. The mammal can be an'embryo, a fetus, or a post natal mammal, e.g., an adult marnimal.
Accordingly, in one aspect, the invention features a method of producing a non-human mammal, e.g., a cloned mammal, e.g., a goat, cow, pig, horse, sheep, llama, camel. The method includes maintaining a mammalian reconstructed embryo, e:g.; a reconstructed embryo wherein the genarne is _g9_ WO OOI26357 ~CT/US~9125710 derived from a somatic cell, in culture until the embryo is in the 2 to- 8 cell stage, transferring the embryo, at the 2 to r cell stage into a recipient mammal, and allowing the reconstructed embryo to develop into a mammal, to thereby produce a mammal.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendarrt of a mammal which developed from the roc~nstructed embryo.
In a preferred ernbodiar~ent, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to 6, the 2 to 4 cell stage of embrycigenesis.
In a preferred emsbodiment, the genome of the reconstructed embryo is derived from: a somatic cell, e.g., a fibroblast or epithelial cell; a genetically engineered somatic cell, e.g., a somatic cell comprising a tra.nsgenic sequence.
in a preferred ert~bodirr~ent, the method further includes mating the ~ 5 mammal which develops from the reconstructed embryo with: a second marnmal;
a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstructed embryo; or a second mamrn:al developed from a reconstructed embryo, or ~ieseended from a mammal which developed from a reconstructed embryo, which was formed from genetic r, ,-n,-;a1 F.-~~ ~3,~ gurn~ a::iTn~~, an anivn_ai of t_h_g catnP gPrinty pPi nr c~,~nE ~e~l ine, a'itawaa a as r=x.,.
which supplied the genetic material for the first mammal. In a preferred embodiment, a first transgenic mammal which develops from the reconstructed embryo can be mated with a second transgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first 25 transgenic mammal.
In a preferred embodiment, the mammal is a male mammal. In other preferred embodiments, the mammal is a female mammal. A female gnammal can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant 35 protein, e.g., a human protein, is recovered from the mammal; a product, e.g., a wo oor~6~s~' ~~r~usg9r~swo protein, e.g., a human protein, is recovered from the milk; urine, hair, blood,. skin or meat of the mammal..
In a preferred embodiment, the mammal is: embryonic; fetal; or, postnatal, e.g.; adult.
. In a preferred embodiment, the genome of theneconstructed embryo is derived from a genetically engineered somatic cell, e.g.,.a transgenic cell or a cell ..
which a nucleic acid has been introduced.
In another aspect; the invention features a rilethod of producing a non-1 o human mammal, e.g., a transgenic mammal, e.g., a l;oat, cow, pig, horse, sheep, llama, camel. The method.includes maintaining a mammalian reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a , genetically engineered somatic cell) in' culture until the embzyo is in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage into a recipient riiammal; and allowing the reconstructed embryo to develop intb a mammal, to thereby produce a transgenic mamrr~al.
In a preferred embodiment, the mammal develops from the reconstructed embryo. In another embodiment, the mammal is a descendant of a mammal which developed from the reconstructed embryo.
In a preferred erribodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to $, the 2 to 6, the 2 to 4 cell stage of embryogenesis.
In a preferred embodiment, the method further includes mating the mammal which develops from the reconstructed embryo with; a second mammal;
a second mammal which develops from a reconstructed embryo or is descended from a mammal which developed from a reconstrut;ted embryo; or a second mammal developed from a reconstructed embryo, or descended from a mammal which developed from a reconstructed embryo, which was formed from genetic material from the same animal, an animal of the same genotype, ~r same cell line, which supplied the genetic material for the first mammal. In a preferred wo oa~2~~s~ ~~~~~~~~ZS°r~a embodiment, a first transgenic nrarrimal which develops from the reconstructed embryo can be mated with a second ttansgenic mammal which developed from a reconstructed embryo and which contains a different transgene that the first transgenic mammal.
In a preferred embodiment, the mammal i5 a male mammal. In other preferred embodiments, the mammal is a female mammal: f~ female mammal ..
can be induced to lactate and milk can be obtained from the mammal.
In a preferred embodirgient: a product, e.g., a protein, e.g., a recombinant protein, e.g., a huriian protein, is recovered from the mammal; a product, e.g., a ~0 protein, e.g., a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the mammal.
In a preferred embodiment, the genome of the genetically engineered somatic cell includes a transgenic sequence.. T'ne transgenic sequence can be amy of a-heterologous transgene9 e.g.9 a human tzan$gene; a,knockout, knockin or.
other event i~rhicl~ disrupts the expression of a mammalian gene; a sequence which encodes a protein; e.g., a human protein; a heterologous promoter; a heterolagous sequence under the control of a promoter, e.g., a caprine promoter.
The transgenic sequence can encode any product of iiiterest.sucli as a protein, polypeptide or peptide.
In a preferred ernbodinaent, the transgenic sequence encodes any of a hormone, an irnmunoglobulin, a plasma protein, and an enzyme. The transgenic sequence can encode any protein whose expression in the transgenic rziamrrial is desired, e.g., any of: 0.-1 proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular supeioxide dismutase, fibrogen, glucocerebrosidase; glutamate decarboxylase, human serum albumin; myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobuliri, lysozye, lactoalbumin, erythrpoietin, tissue plasminogen activator; human growth factor, antithrombin III, insulin, prolactin, arid al-antitrypsin.
In a preferred embodiment, the transgenic sequence encades a human protein.
~2~.

W~.00/26357 ~~TlL1S99125710 In a preferred embodiment,, the transgenic sequence is urgder the control of a promoter, e.g., a caprine or heterologous prombter. The promoter-can be a tissue-specific promoter. The tissue specific promoter can be any of: milk-specific promoters; blood-specific promoters; muscle-specific prom~ters;
neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can: be, e.g., any of: a casein ..
prom~ter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In a preferred embodiment, a.nucleie acid can be introduced into the gename of the genetically engineered somatic cell. The nucleic acid can be any of a heterologous transgene, e.g., a human transger~e; a knockout, knockin or other event. which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine or heterologous promoter. The nucleic acid sequence can encode any product of interest such as a protein, polypeptide or peptide. .
in a preferred embodiment, the nucleic acid encodes any of a hormone, an immunoglobulin, a plasma protein, and an enzyme. The nucleic acid sequence can encode any protein whose expression in the tra:nsgenic mammal is desired, 2o e.g., any of: a-i proteinase inhilsitor, W tcaiine phosophotase, a:ngiugeyu, extracellular superoxide dismutase, fibrogen, gluco~cer~brosidase, glutarraate decarboxylase, human serum albumin, myelin basic pr~tein, proirisulin, si~luble CI?4, lactoferrin, lactoglobulin, lysozyme, 3actoalbumin, erythrpoietin, tissue plasrninogen activator, human growth factor, antithrombin III, ansulin, prolactin, 25 and ocI-antitrypsin.
In a preferred embodiment, the nucleic acid sequence encodes a human protein.
In a preferred embodiment, the nucleic acid sequence is under the control of a promoter, e.g., a.caprine or heteroiogous pron:~oter. T'he.promoter carp be a 30- tissue-specific promoter: The tissue specific promoter care be any of:
milk-WO 00/26357 P~'rlUS99l25Tt0 specific promoters; blood~specific promoters; muscle-specific promoters;
neural-.specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of: a casein promoter, a beta lactoglobulin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect, the invention features a method of producing a cloned .
goat. The method includes maintaining a capriile reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a capririe somatic call) in culture until the embryo is. in the 2 to 8 cell stage, transferring the embryo at the 2 to 8 cell stage unto a recipient goat, and allowing the reconstructed embryo to develop into a goat, to thereby produce a goat.
In a preferred embodiment, the goat is: embryonic; fetal; or, postnatal, e.g:, adult.
In a preferred embodiment, the goat develops from the reconstructed embryo. In another embodiynent, the goat is a descendant of a goat which developed frown the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained in culture until the embryo is in the 2 to 8, the 2 to f, the 2 to 4 cell stage of .
er!~brj ogenPC7C, , In a preferred embodiment, the genome of the reconstructed embryo is derived from: a caprine somatic cell, e.g., a fibroblast or epithelial cell; a genetically engineered caprine somatic cell, e,g., the genome 'of the caprine .
somatic cell comprises a.transgenic sequence or a nucleic acid has been introduced into tlae genome of the somatic cell.
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo with: a second goat;-a second goat which develops from a reconstructed embryo or is descended from a goat which developed from a reconstructed embryo; or a second goat developed from a 30. reconstr acted embryo, or descended from a goat which developed from a WD OOi263S7 ~c~rms~~~xs~ro reconstructed embryo, which was formed from. genetic material from the same animal, an animal of the same genotype, or same cell. Iine, vahich supplied the genetic material for the f rst goat. In a preferred embodiment, a- first transgenic goat which develops from the reconstructed embryo can be mated with a,sec~ind transgenic goat which developed from a reconstructed embryo and which contains a different transgene that the first transgenic; goat.
In a preferred embodiment, the goat is a male. goat. In other preferred embodiments; the goat is a female goat. A female goat can be induced to lactate and milk can be obtained from the goat.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human protein, is recovered from the goat; a pioduct, e.g., a protein; e.g,, a human protein, is recovered from the milk, urine, hair, blood, skin or meat of the goat. .
In another aspect, the invention features a method of producing a transgeriic goat. T'he method includes maintaining a. caprine reconstructed embryo (e.g., a reconstructed embryo wherein its genome is derived from a genetically engineered somatic cell) in culture until 'the embryo is in the ~
to 8 cell stage; transferring the embryo at the 2 to 8 cell stage into a recipient goat, and . allowing the reconstructed embryo to develop into a. goat, to thereby.
produce a ~n~genic goat.
In a preferred embodiment, the goat is: embryonic; fetal; or, postnatal, e.g:, adult.
In a preferred embodiment, the goat develops from the reconstructed embryo. Iri another embodiment, the goat is a descendant of a goat which developed from the reconstructed embryo.
In a preferred embodiment, the reconstructed embryo is maintained im culture until the embryo is in the 2 to 8, the 2 to b, the 2,to 4 cell stage of embryogenesis. - .
_45-WO flfl~26357 ~C't'/CTS99125?t0 In a preferred embodiment, the genome of the reconstructed embryo is derived from a somatic cell, e.g., a fibroblast or epithelial cell.
In a preferred embodiment, the method further includes mating the goat which develops from the reconstructed embryo with: a second goat; a second goat which develops from a reconstructed embryo or is descended from a goat which developed from a reconstructed embryo;.or a second goat developed from a reconstructed embryo, ar descended from a gaat which developed from a reconstructed embryri, which was formed from genetic material from the same animal, an animal of the same genotype, or same cell Line, which supplied the 1 o : genetic material for the fzrst goat. In a preferred embodiment, a first transgenic goat which develops from the reconstructed embryo can be mated with a second transgenic goat which developed from a reconstructed embryo and which contains a different transgene that the first transgenic goat.
In a preferred embc~dirnent, the goat is a anale goat. In other preferred embodiments, the goat is a female goat. A female goat can be induced to lactate and milk can be obtained from the goat.
In a preferred embodiment: a product, e.g., a protein, e.g., a recombinant protein, e.g., a human proteixi, is recovered from the goat; a product, e.g., a protein, e.g., a human lirotein, is recovered from the milk, urine, hair, blood, skin w or iiieat.of iii2 goat.
In .a preferred embodiment, the genome of the genetically engineered somatic cell includes a transgenic sequence. The transgenic sequence cari be any of a heterologous traxisgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protean, e.g., a human protein; a heterologous promoter; a heterologous sequence under the control of a promoter, e.g., a caprine or heterologous promoter: the transgenic sequence can encode any product ~f interest such as a protein, polypeptide or peptide. .
In a preferred embodiment, the transgenic sequence encodes any of a hormone, an immunoglobulin, a plasma protein, and an enzyme. _ 'The transgenic a~6_ WO 00!26357 PC'fItJS99125710 sequence can encode any protein whose expression in the transgenic mammal is . desired, e.g., any of: a-I proteinase inhibitor, alkaline phosphotase, angiogenin, extracellular superoxide dismutase, ~brogen, glucoce;rebrosidase; glutamate _ decarboxylase, human serum albumin, myelin basic protein, proinsulin, soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin, erythzpoietin, tissue plasminogen activator, human growth .factor, antithrombin II I, insulin, prolactin, and a.l-antitrypsin.
In a preferred embodiment, the transgenic sequence encodes a human protein.
In a preferred embodiment, the transgenic sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any of milk-specific promoters; blood-specific promoters; muscle;-specific promoters;
neural-specific promoters; skin=specife promoters; lair-specific promoters; and urine-specific pFOmoters. The milk-specific promoter can be,. e.g., any of: a casein promoter, a beta Iactoglobulin promoter, a ~vvhey acid protein promoter.and a lactalbumin promoter.
In a preferred embodiment, a nucleic acid has been introduced into the genome of the genetically engineered somatic cell. °I"he nucleic acid sequence can be any:of a heterologous transgene, e.g., a human transgene; a knockout, knockin or other event which disrupts the expression of a mammalian gene; a sequence which encodes a protein, e.g:, a human protein; a heterologous promoter; a heterologous. sequence under the control of a promoter, e.g., a caprine or heterologous promoter. The txansge~ic sequence .can encode and product of interest such as a protein, poIypeptide or peptide.
In a preferred embodiment, the nucleic acid encodes any of a hormone, an immurioglobulin, a plasma protein, and an enzyme. The nucleic acid sequence can encode any protein whose expression in the transgenic mammal is desired, e.g., any of: a-1 proteinase inhibitor, alkaline phospliotase, angiogenin, extracellular superoxide dismutase, fibrogen, glucocerebrosidase, glutamate WO 00/26357 ~'~T/tJ~99/z57t~
decarboxylase, human serum albumin, myelin basic protein; proinsulin, soluble CD4, lactoferrin, lactoglobulin, Iysozyrne, lactoalbumin, erythrpoietin, tissue plasminogen activator, human growth factor, antithrombin III, insulin, prolactin, and al-antitrypsin.
In a preferred embodiment, the nucleic .acid sequence encodes a lawman protein. ..
In a preferred embodiment, the nucleic acid sequence is under the control of a promoter, e.g., a caprine or heterologous promoter. The promoter can be a tissue-specific promoter. The tissue specific promoter can be any ofo milk-specific promoters; blood-specific promoters; muscle-specific promoters;
neural-specific promoters; skin-specific promoters; hair-specific promoters; and urine-specific promoters. The milk-specific promoter can be, e.g., any of a casein promoter, a beta lactoglobuiin promoter, a whey acid protein promoter and a lactalbumin promoter.
In another aspect; the invention features a kit. The kit includes a reconstructed embryo which is in the 2 to 8 cell stage. In a preferred embodiment, the kit further includes instructions for producing a mammal, e.g., an embryonic, fetal or postnatal mammal.
In another aspect, the invention features a kit which includes, a later stage embryo, e.g., an embryo after the 8 cell stage, or a fetus, obtained, e.g., by the methods described herein.
As used herein, the term "functional enucleation'rrefers to a process of rendering the endogenous genome of a cell; e.g., an oocyte, incapable of functioning, e.g., replicating and/or synthesizing DNA. Such an oocyte is referred to herein as a "functionally enucleated oocyte".

!V~ 00/26357 ~CTJUS99I25710 The terms protein, polypeptide and peptide a:re used interchangeably herein.
As used, herein, the term "transgenio sequence" refers to a nucleic acid sequence (e.g., encoding one of rnore human proteins), which is inserted by artifice into a cell. The transgenic sequence, also referred to herein as a transgene, becomes part of the genome of an animal which develops in whole or in part from that cell. In embodiments of the invention, the transgenic sequence is integrated into the chromosomal genome. If the firansgenic sequence is integrated into the genome it results, merely by virhde of its insertion, in a change in the nucleic acid sequence of the genome into which it is inserted. A
transgenic sequence can be partly or entirely species-heterologous, i.e., the transgenic sequence, or a portion thereof, can be from a species which is different from the cell into which it is introduced. A transgenic sequence can be partly or entirely species-homologous, i.e., the transgenic sequence, or a portion thereof, 'can be from the same species as is the cell into. which it is introduced. If a trarisgenic.
sequence is homologous (in tlae sequence sense or in the species-homologous sense) to an endogenous gene of the cell into which it is introduced, then the transgenic sequence, preferably, has one or more of the following characteristics:
. it is designed for insertion, or is inserted, into the ce;Il's genorrne iii such a way as to alter the sequence of the genome of the cell into which it is inserted (e~g., it is inserted at a location which differs from that of the endogenous gene or its unsertion results in a change in the sequence of the endogenous endogenous gene); it includes a mutation, e.g., a mutation which results in misexpression of the transgenic sequence; by virtue of its insertion, i~t can result in misexpression of the gene into which it is inserted, e.g.~ the insertion can result in a knockout of the gene into which it is inserted. A transgenic sequence can include one or more transcriptional regulatory sequences and any other :nucleic acid sequences, such as introns, that may be necessary for a desired level or pattern of expression of a 3~ selected nucleic acid, all operably linked .to the selected nucleic acid. -'file WO OQ1263S7 PC'~'/US391257~~
transgenic sequence can include an enhancer sequence and or sequences urhich allow for secretion.
The terms ''reconstructed embryo", "reconstituted embryo", "nuclear transfer unit" and "nuclear transfer embryo" are used interchangeably herein.
As used herein, 'the term "normal goat" refers to a goat which did not develop 'from a reconstructed. embry~.
1Q . A "naturally derived oocyte" is one which is allowed to reach a selected stage, e.g., metaphase II or more preferably telophase, by culturing under natural conditions, e.g., in vivo. The term "natural, conditions" means the absence of treatment of the oocyte with exogenously added chemicals, e.g:, ethanol, to affect . the stage of meiosis. In preferred embodiments, a naturally matured preparation can include metaphase sI, telopl-Aase or both.stages. The inventors have discovered that naturally matured oocytes are preferable to those which have been chemically induced.
Other features and advantages of the invention will be apparent from the ~n following description and from the claims.
Det~dled Desc~°ipti~pa ~f'fhe dsaveeati~ra Sources of Somatio Cyenomes:
Somatic Cells . .
Somatic cells can supply the genome for producing a reconstructed embryo in the methods described herein. 'fhe term "somatic cell", as used herein, refers to a differentiated cell. The cell can be a somatic cell or a cell that is committed to a somatic cell lineage. Alternatively, any of the methods and WO 00/26357' ~CTlUS99/25'710 animals described herein can utilize a diploid stem cell that gives rise to a germ cell in order to supply the genome for producing a reconstructed embryo.
The somatic cell can be from an animal or from a cell culture. If taken from an animal, the anirilal can be at any stage of development, e.g.; an embryo, a . fetus or an adult. Embryonic cells .are preferred. Embryonic cells can include embryonic stem cells as well as embryonic cells committed to a somatic cell -lineage: ySuch cells can be obtained from the endoderm, mesoderm or ectoderm of the embryo. Preferably; the erzabryonic cells are committed to somatic cell lineage. Embryonic cells committed to a somatic cell lineage refer to cells isolated on or after day 10 of embryogenesis. I-Iowe;ver, cells can be obtained prior to day ten of embryogenesis. If a cell line is used as, a source of a chromosomal genome, primary cells are preferred. The term "primary cell line"
as used herein includes primary cell lines as well as primary-derived cell lines.
Suitable somatic cells include fibroblasts (e.g., primary fibroblasts, e.g., embryonic primary fibroblasts), muscle cells (e.g., myocytes), cumulus cells, .
neural cells; and mammary calls. ~ther suitable cells include hepatocytes and pancreatic islets. Preferably, the somatic cell is an embryonic somatic cell, e.g., a cell isolated on or after day 1 U of embryogenesis: 'The genome 'of the somatic cells can be the naturally occurring genome, e.g:, fmr the production of cloned . mammals, or the,genome can be genetically altered to comprise a transgenic sequence, e:g.; for the production of transgenic cloned mammals.
Somatic cells can be obtained by, for example, dissociation of tissue, e.g., by mechanical (e.g., chopping, mincing) or enzymatic means (e.g., trypsinization) to obtain a cell suspension and then by culturing the cells until a confluent monolayer is obtained. The somatic cells can then be harvested and prepared for cryopreservation, or maintained as a stock culture. The isolation of caprine somatic cells, e.g., fibrobIasts; is described herein.
The somatic cell can be a quiescent or non-quiescent somatic cell. "Non-quiescent", as used herein, refers to a cell in mitotic cell cycle. The mitotic cell 30. cycle has four distinct phases, G" 8, GZ and M. The beginning event in the cell W~ UO/26357 ~C't'JIJS991257t0 cycle, called START, takes place. during the G, phase. "START" as used herein , refers to early G, stage of the cell cycle prior to the commitment of a cell to proceeding through the cell cycle. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1~
up to 11 hours after a cell enters the G, stage, the cell is considered prior to START.
The decision as to whether tlye cell will undergo, another cell cycle is made at START. Once the cell has passed thr~ugh START, it passes through the remainder of the G, phase (i.e., the pre-I7NA synthesis stage). The S phase is the DNA synthesis stage, which is followed by the GZ phase, the stage between synthesis and mitosis. Mitosis. takes place during the IvI phase. If at, START, the cell does not undergo another cell cycle, the cell becomes quiescent. In addition, a cell can be induced to exit the. cell cycle_and become quiescent. A
"quiescent"
cell, also referred to as a cell ire Go phase, refers to a cell which is not in any of the four .phases of the cell cycle. Preferably; the somatic cell is a cell in the Gophase or the G, phase of the mitotic cell cycle.
Using donor somatic cells at certain phases-of the. cell cycle, e.g., Ga ~r G, phase, can ~ltow for synchronization between the ~ocyte and the genome of the somatic cell. For example, reconstruction of an oocyte in metaphase II by introduction of a nucleus of a somatic cell in Go or G,, e.g., by simultaneous activation and fusi~n, can animic the events occurring dui"ing fertilization.
13y way of another example, an oocyie in teloplaase II fused, e.g., by simultaneous activation and fusion, .with the gen~me of a somatic cell in G, prior t~
START, provides a synchronizatioai of cell cycle between the oocyte and donor nuclei.
Methods of deteritiining which phase of the cell cycle a cell is in are known. For example, as described below im the Examples, various markers are present at different stages of the cell .cycle. Such markers can include cyclins I~
1, 2, 3 and proliferating cell nuclear antigen (PCNA) for G" and BrDu to detect DNA synthetic activity. In addition, cells can be induced to enter the Go stage by culturing the cells on serum-deprived medium. Alternatively, cells in Go stage can be induced t~ enter the cell cycle, i.e., at G, stage, by serum activation.

50409-13D(S) The-donor cells can be obtained from a mammal, e.g., an embryonic,, fetal or adult mammal, or from a culture system, e.g., a synchronous culture system.
For example, the donor cell can be selected from a culture system which contains at least a majority of donor cells (e.g., 50%, 55%, 60%; 65%, 70%, 75%, 80%, . '85%, 90% or more) in a specif c stage of the mitotic.cell .cycle.
Sources of Genetically Engineered Somatic Cells:
Trans~enic Mammals Methods for generating non-human transgenic mammals which can be used as a source of somatic cells in the invention are known in the art. Such methods cari involve introducing DNA constructs into the germ line of a mammal to make a transgenic mammal. For example, one or several copies of the construct may. be incorporated intoahe genome of a mammalian embryo by standard transgenic techniques. ' Although goats are a preferred source of genetically engineered somatic cells, other non-human mammals can be used. Preferred non-human mammals are ruminants, e,g., cows, sheep, camels or goats. .Goats of Swiss origin, e.g., the Alpine, Saanen and Toggenburg breed goats, are useful in the methods described herein. Additional examples of preferred non-human animals include oxen, horses, llamas, and pigs.' The mammal used as the source of genetically engineered cells wilt depend on the transgenic mammal to be obtained by the methods of the invention as, by way of example, a goat genome should be introduced, into a goat functionally enucleated ooeyte.
Preferably, the somatic cells for use in the invention are obtained from a 25, transgenic goat. Methods of producing transgenic goats are known in the art. For example, a transgene~can be introduced into the germline of a goat by microinjection.as described, for example; in Ebert et al. (1994) BiolTechnology 12:699 .
Other transgenic non-human animals to be used as a source of genetically . engineered somatic cells can be produced by introducing a transgene into the W~ 00/26357 ~CT/f.JS~9/25730 germline of the non-human animal. Embryonal_ target cells at various developmental stages can be uscd to introduce transgenes. Different methods are , , used depending on the stage of development of the embryonal target cell. The specific Iine(s) of any animal used to practice this-invention are selected for general good health, good embryo yields, good pronuclear visibility in the.
embryo, and good rcproductive fitness. In addition, the haplotype is a significant factor.
Transfected Cell Lines Genetically engineered somatic cells for use in the invention can be obtained from a cell line into which a nucleic acid of.interest, e.g., a,nucleic acid which encodes a protein, leas been introduced.
A construct can be introduced into a cell via conventional transformation or transfection techniques. As used herein, the terms "transfection" and "transformation" include a variety of techniques for introducing a trarisgenic sequence into a host cell, including calciuhZ_phosplaate or calcium chloride co-precipitation, DEAF-dextrane-mediated transfec~ion, lipofection, or electroporation. In addition, biological vectors; e.g., viral vectors can be used as described below: Suitable methods for transf~rming or trarisfecting host cells can Ltd be foiiud in ~aWbrvva ei ai., i vlci,ului viGi'Ii'g:.ri ~.T,uwYCasi~Y°y wi iiiP.s'2i 2nd L'~.., Cold Spring Herb~r Labos-catory, (Cold Spxing Harbor Laboratory Press, Cold Spring Harbox, NY, I989), and other suitable laboratory manuals.
Two useful approaches are eIectroporation and lipofection. Brief examples of each are described below.
The DNA construct can be stably introduced into a donor somatic cell Line by electroporation using the following protocol; somatic cells, e.g., fibroblasts, e.g., embryonic fibroblasts, ate resuspended in PES at about 4 x 106 cells/rral.
Fifty rnicorgrams of linearized DNA is added to the 0.5 ml cell suspension, and the suspension is placed in a 0.~ can electrode.gap cuvette (Biorad).
Electroporation is performed using a Biorad Gene Pulser electroporator with a 330 volt pulse at 2S mA,1000 rnicroFarad and.infinite resistance. If the Dl~IA
construct contains a Neomyocin resistance gene for selection, neomyocin resistant clones are selected following incubation with 3S0 micio_gramlml of 6418 (GibcoBRL) for 1 S days.
The DNA construct can be stably introduced into a donor somatic cell line by lipofection using a protocol such as the following: about 2 x 105 cells are -.
plated. into a 3.S cmiameter well and transfected with 2 rnicrograrns of linearized DNA using LapfectAMINET"" (GibcoBR.L). Forty-eight, hours after transfection, the cells are split 1:1000 and 1:5000 and, if the DNA construct contains a neornyosin resistance gene for selection,, G4i8 is added to a final.concentration of 0.35 mg/ml. Neomyocin resistant clones are isolated and.expanded for cyropreservation as well as nuclear transfer.
Tissue-Specific Expression of Proteins It is often desirable to express a protein, e.g:, a heterologous protein, in a specific tissue or fluid, e.g., the milk, of a transgenic animal: The heterologous protein can be recovered from the tissue or fluid in which it is.expressed.
For example; it is often desirable to express, the heterolol;ous protein in milk.
Methods for producing a heterologous protein under the control of a milk specific promoter are described below. In addition, other tissue-specific promoters, as well as, other regulatory elements, e.g., signal sequences and sequence which enhance secretion of non-secreted proteins, are descy~ibed below.
lViilk Specific Promoters I3seful transcriptional promoters are those promoters that are preferentially activated in mammary epithelial cells,. including promoters that control the genes encoding milk proteins such as caseins, beta lactoglobulin (Clark et al., (1989) Bio/Technology 7: 487-492), whey acid protein (Gordon et al. (1987) BiolTechnology 5: 1183-1187); and lactalbumin (Soulier et al., (1992) WO ~0/26357 1'CTliJS99l257t0 FEBS Letfs. 297: ~. Casein promoters may be derived from the alpha, beta, gamma or kappa casein genes of any mammalian species; a preferred promoter is derived from the goat beta casein gene (DiTuilio, (1992) Bio/Technology 10:74-77). Milk-specific protein promoter or the promoters that are specifically activated in mammary tissue can be derived from cDNA or genomic sequences.
Preferably, they are genoaxaic in origin. ..
DNA sequence information is available for the mammary gland specific genes listed above; in at least one, and often in several organisms. See, e.g., Richards et al., J. I3iol. Claem. 256, 526-532 (1981) (oc-lactalbumin rat);
Campbell et al:, Nucleic Acids Res. i2; 8685-8697 (1984) (rat WAP); Jones et al., J.
Biol.
Chem. 260, 7042-7050 (1985) {rat ~i-casein); Yu-Lea ~ Rosen, J. Biol. Chem.
258, 10794-10804 (1983) (racy-casein); l'-iall, Biochem. J. 242, 735-74.2 (1987) ( a-Iactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984) (bovine ~,sl and K casein cDNAs); (rorodetsky et al., ~iene 66, 87-96 (1988) (bovine (3 casein); Alexander et aL, Irur. J. Biochem. I789 395-401 (2988) (Bovine x casein);
Brignon et al., FEBS Left. 188, 48-55 {1977) (bovine a.S2 casein); Jamieson et al:, Gene 6l, 85-90 (1987), Ivanov et al., Biol. Chem. Hloppe-Seyler 369, 425-{1988), Alexander et al., Nucleic Acids hes. 17, 6739 (1989) (bovine (3 lactoglobulin)p'Vilotte et al., Biochimie 69, 609-620 (1987) (bovine a-zo iactaibumin). The structure and ~nciior~ of the vari~ii~s mili~ proieira genes are reviewed by Merrier ~ '~Iilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated by reference in its entirety for all purposes). If additional flanking sequence are useful in optimizing expression of the heterologous protein, such sequences can be cloned using the existing sequences as probes: IViammary-gland specific regulatory sequences from different organisms can be obtained by screening libraries from such organisms using known cognate nucleotide sequences, or antibodies to cognate proteins as probes.
Signal Sequences _gg_ W~ 00/26357 P~T/US99/25714 Useful signal sequences are milk~specific signal sequences or other signal sequences which result iri the secretion of eukaryotic or prokaryotic proteins.
Preferably, the signal sequence is selected from milk-specific signal sequences, i.e., it is from a gene which encodes a product secreted into milk. Most preferably, the milk-specific signal sequence is related to the milk-specific promoter used in the construct, which are described below. The size of the signal ..
sequence is not critical. All that is required is that the sequence be of a suff cient size to effect secretion of the desired recombinant protein, e.g., in the mammary tissue. For example, signal sequences from genes coding for caseins, e.g., alpha, 1 o beta, gamma or kappa caseins, beta lactoglobulin, whey acid protein, and lactalbumin can be used: A preferred signal sequence is the goat ~i-casein signal sequence.
Signal sequences from othei secreted proteins, e.g.; proteins secreted by kidney cells, pancreatic cells or liver Bells, can also be used. Preferably, the signal sequence results in the secretion of proteins into, for example, urine or blood.
Amino-Terminal Regions of Secreted Proteins A non-secreted protein can also be modified in such a manner that it is 2u secreted such as by inclusion in the protein to be secreted of iii or pait of the coding sequence of a protein which is normally secreted. Preferably the entire sequence of the protein vrhich is normally secreted is not included in the sequence of the protein but rather only a sufficient portion of the amino terminal end of the protein which is normally secreted to result in secretion of the protein. For example, a protein which is not normally secreted is fused (usually at its amino terminal end) to an amino terminal portion of a protein which is normally secreted.
In one aspect, the protein which is normally secreted is a protein which is . normally secreted in milk. Such proteins include proteins secreted by mammary 3o epithelial cells, milk proteins such as caseins, beta lactoglobulin, whey acid _57_ W~ 00/26357 PC'r/IJ~991~S7d0 protein , and lactalburrain. Casein proteins inctude alpha, beta, gamma or kappa .
casein genes of any rndmmalian species. A preferred protein is beta casein;
e.g., goat beta casein. 'fhe sequences which encode the secreted protein can be derived from either cl_7I~A or genomic sequences:. Preferably, they are genomic in origin, and include one or more iritrons.
Other °Tissue-Specific Promoters Other tissue-specific promoters which provide expression in a particular tissue can be used. Tissue specific promoters are promoters which are expressed 1o more strongly in a particular tas5ue than in others. 'Tissue specific promoters are often expressed essentially exclusively in the. specific tissue.
Tissue-specific promoters which can be used include: a tleural-specific promoter, e.g., nestin, ~7nt-1, fax-1, Engrailed-l, Engrailed-~, Sonic hedgehog; a liver=specific proyraoter, e.g., albumin, alpha-1 antirypsin; a muscle-specific promoter, e.g., Irlyogenin, actin, ~yd0; myosin; an oocyte specific promoter, e.g., ZP1, ZP2, ZP3; a testes-specifac promoter, ~.g., protamiri, fertiliri, synaptonernal c~mplex protein-1; a blood-specific promoter, e.g., globulin, GATA-l, porphobilinogen deaminase; a lung-specific promoter, e.g., surfactant protein C; a skim- or wool-specific promoter, e.g.,.lceratin, elastin;
endotheliurri-specific promoters, e.g.,'fie-i, q°ie-2; and a bone-specify promoter, e.g., BI~P.
In additioai, general promoters can be used for expression in several tissues. Examples of general promoters include (3-actin, ROSA-21, PCr~, F~S, c-.
myc, Jun-A, and Jun-l~.
OhIA Constructs A chssette which encodes a heterologous protein can be assembled as a construct which includes a promoter for a specific tissue, e.g., for marrlrnary epithelial cells, e.g., a casein promoter, e.g., a goat beta casein promoter, a milk-specific signal sequence, e.g., a casein signal sequence,.e.g., a (3-casein signal 3o sequence, arid a I)leTA encoding the heterologous protein.
_5g_ VVO.00/26357 PCTfLJS99125710 The construct can also include a 3' untcanslated region downstream of the DNA sequence coding for the non-secreted protein. Such regions can stabilize the RNA transcript of the expression system and thus increases the yield of desired protein from the expression system. Among the 3' uritranslated regions useful in the constructs for use in the invention are sequences that provide a poly A signal. Such sequences may be derived, e.g.,. from the SV40 small t antigen, the casein 3'-untranslated region or other 3' untranslated sequences vsrell known in the art. In one aspect, the 3' unixanslated region is dc;rived from a milk specific protein. The length of the 3' untranslated region is not critacal but the stabilizing effect of its poly A transcript appears important in stabilizing the RNA of the expression sequence.
Optionally, the construct can include a 5' untranslated regiom between the promoter and the DNA sequence encoding the signal sequence. Such untranslated regions can be from the same control region from Which promoter is is taken or can be from a different gene, e_g., they may be derived from other synthetic; semi-synthetic or natural sources. Again their specific-length is not critical, however, they appear to be useful in improving the level of expression.
The construct can also include about 10%, 20%; 30%, ~r more of the N
terminal coding region of a gene preferentially expressed in mammary epithelial 2~7 cells. nor example, the hi-terminal coding region can correspond to the,prornoter used; e.g., a goat ~3-casein N-terminal coding region.
The construct can be prepared using methods known .in the art. 'I""he constiucf can be prepared as part of a larger plasmicf. Such preparation allows the clotting and selection of the correct constructions in an efficient manner.
The 25 construct can be located between convenient restriction sites on the plasmid so that they can be easily isolated from the remaining plasmid sequences for incorporation into the desired mammal.
Heterologous Proteins _59.

WO OOI2635? PCTltJS99/2~7H0 Transgenic sequences encoding heterologous proteins can be introduced into the germline of anon-human mammal or can be transfected into a cell line to provide a source of genetically engineered soraiatic cells as described above.
The protein can be a complex or muitiirieric protein, e.g., a homo- or heteromultimer, e.g., proteins ~rhich naturally occur as homo- or heteromultimers, e.g., homo- or hetero- dimers, trimers or tetramers. The protein -.
can be a protein which is processed by removal, e.g., cleavage, of N-terminus, C-terminus or internal fragments. Even conipIex proteins can be expressed in active form. Protein encoding sequences which cah be introduced into the genome of mammal, e.g., goals, include glycoproteins; neuropeptides, immunoglobulins, enzymes, peptides and hormones. The protein may be a naturally occurring protein or a recombinant protein, e.g., a fragmaent, fusion protein, e.g., an immunoglogulin fusion protein, or mutien. It may be human or non-human in origin. The heterologous protein naay be a potential therapeutic ~r Z5 pharmaceutical agent such as, but nod limited to; alpha-1 proteinase inhsbitor, alpha-1 atltitrypsine, alkaline phosphatase, angiogenin, antithrombin III, any of the blood clotting factors including Factor VIII, Factor IX, and Factor ~C
chitinase, erythropoietin, extracellutar superoxide dismutase, fibrinogen, glucocerebrosidase, glutamate decarboxylase, human growth factor, human serum 20 albumin, immunoglobulin, insulin, myelin basic protein, proinsulin, prolactin, soluble CD4 ox a component ~r complex thereof, lactofernn, laetoglobulin, lysozyme, lactalbumin, tissue plasminogen activator or a variant thereof Immunoglobulins are particulafly prefered heterologous protiex<s.
Examples of immunoglobulins include IgA, IgCi, IgE, Igl~, chimeric antibodies, 25 humanized antibodies, recombinant antibodies, single chain antibodies and antibody-protein fusions.
Nucleotide sequence information is available for several of the genes encoding the heterologous proteins listed above, in at least one, and often in several organisms. See e.g., I_,ong et aI. (I984) Ba~chen~. 23(2I):4828-4837 30 (aplha-1 antitry~sin); Il~itchell et al. (1986) 1'r~Z, rlatl. .Acted. Sci RIS~4 83:7182-50409-13D(S) 7186 (alkaline phosphatase); Schneider et al. (1.988)~EMBOJ. 7(13):4151-4156 (angiogenin); Bock et ~al..(1988) Biochem. 27(16):6171-6178 (antithrombin III);
Olds et al. (1991) Br. J. Haematol. 78(3):408-413 (antithrombiri.III); Lin et al:
(1985) Proc.~Natl. Acad. Sci. USA~82(22):7580-7584 (erythropoeitin); U.S.
Patent No. 5,614,184 (erythropoietin);~Horowitz et al. (1989) Genomics 4(1):87 96 (glucocerebrosidase); Kelly et al: (1992) Ann. Hum. Genet. 56(3):255-265 (glutamte decarboxylase); U.S: Patent No. 5,707,828 (human serum albumin); .
U.S. Patent No. 5,652,352 (human serum albumin); Lawn et al. (1981) Nucleic Acid Res. 9(22):6103-6114 (human serum albumin); Kamholz et al. (1,986) Prot.
Natl. Acad. Sci. USA 83(13):4962-4966 (myelin basic.piotein); Hiraoka et al.
(1991) Mol. Cell Endocrinol. 75(1):71-80 (prolactin); U.S: Patent No:
$,571,896 (lactoferrin); Pennica et al. (1983) Natura 301 (5897):214-221 (tissue plasminogen activator); Sarafanov,et al. (1995)Mol. Biol. 29:161-165.

Ooc es . Oocytes for use in the invention include oocytes in metaphase II stage of meiotic cell division, e.g., oocytes arrested in metaphase II, and telophase stage of meiotic division, e.g., telophase I or telophase IL -Oocytes in metaphase II
. contain one polar body, whereas oocytes in telophase can be identified based on the presence of a protrusion of the plasma membrane. from the second.polar body up to the formation of a second polar body. In addition, oocytes in metaphase II
can be distinguished from oocytes in telophase 1I based on biochemical and/or developmental distinctions. For example, oocytes in metaphase Ih can be in an arrested state, whereas oocytes in telophase are in an activated state.
Preferably, the oocyte is a~caprine oocyte.
Occytes can be obtained at various times during a goat's reproductive cycle. For example, at given times during the reproductive cycle, a significant percentage of the oocytes, e.g., about 55%, 60%, 65%, 70%, 75%, 80% or more, . ~ . are oocytes in telophase. 'These oocytes are naturally matured oocytes.-1n -61- . ~ .

W~ 00/26357 P~'d'IIJS99/25710 addition, oocytes at various stages of the cell cycle can be obtained and then induced in vitro to enter a particular stage of meiosis. For example, oocytes .
cultured on serum-starved medium become arrested in metaphase. In addition, arrested oocytes can be induced to enter telopliase by seryn activation. Thus, .
oocytes in telophase can be easily obtained for use in the invention:
Oocytes can be matured in vitro before they~are rised to form a reconstructed embryo. 'I his process usually requires collecting imrnatiare oocytes from mammalian ovaries, c.g., a caprine ovary, and maturing the oocyte in a medium prior to enucleation until the oocyte reaches the desired meiotic stage, e.g., metaphase or telophase. In addition, ~ocytes that have been matured in vavo can be used to f~rm a t-econsta-ucted embryo.
Oocytes can be collected, from a female mammal during superovulation.
Briefly, oocytes, e.g., caprine oocytes, can be recovered surgically by hushing the oocytes from the oviduct of the female donor. Methods of inducing 15- superovitiation in goats and the collection of caprine oocytes is described herein.
Preferably, the meiotic stage of the oocyte, e.g., gnetaphase,Il or telophase Il, correlates to the stage of the cell cycle of the donor somatic cell. The correlation betvi~een the meiotic stage of tlae oocyte and the mitotic stage mf the cell cycle of the donor somatic cell is referred to herein as "synchronization". For 2v °v~'llinple re~v::ut:~.~.:a::'~: of an aorac°.wt_P in IvvPta»rh~y IT by iI'ti_-a_'Qdl~Ctio%1 ~f a Xlii~l~idS
of a somatic cell in Ga or (.~" e.g:, by simultaneous activation and fusion9 can mimic the events occurring during fertilization. By way of another example, an oocyte in telophase fused, e.g., by simultaneous activation and fusion, with the genome ~f a somatic cell in G~ prior to START, provides a synchronization 25 between the.oocyte and the donor nuclei.
Functional Enucleation 'The donor oocyte, e.g., capaine oocyte, should be functionally enucleated such that the endogenous genome of the oocyte is incapable of functioning, e.g., 30 replicating or _g2_ WO 00/26357 PC."T~tJS99/25710 synthesizing DNA. Methods of functionally enuclearing an oocyte include:
removing the genome from the oocyte (i.e., enucleation), e.g., such that the ' oocyte is devoid of nuclear genome; inactivating DhIA within the oocyte, e.g., by .
irradiation (e.g., by X-ray irradiation, or laser irradiation); chemical .inactivation, or the like.
Enucleation One method of rendering the genome of an oocyte incapable of functioning is to remove the genome from the oocyte (i.e., enucleation). A
micropipette or needle can be inserted into the zona pellicuda in order to remove nuclear material from an oocyte. For example, metaphase II stage oocytes which have one polar body can be enucleate~ with a micropipette by aspirating the first polar body and adjacent cytoplasm surrounding the polar body, e.g., approximately 20%, 30%, 40%, 50%, 60% of the cytoplasm, which presumably contains the metaphase plate. 'Telphase stage oocytes which have two polar bodies can be enucleated with a micropipette orneedle by removing the second polar body and surrounding cytoplasm, e.g., approximately 5%, 10%, 20°f°, 30%, 40%, 50%, 60% of cytoplasm: Specifically, oocytes in telophase stage can be enucleated..at any point from the presence of a protrusion in the plasma membrane ~~ from tlZe se~'n,~d p~nla,: 1,'!v'~y'vip to thra fnrmot;nn of fhe se~::rod pf~'l3r.bc~dy 'T'hy.C.
a. a vvu aasv vmaswav , as used herein, oocytes which demonstrate a protn~sion in the plasma membrane, usually with a spindle abutted to it, up to extrusion of the second polar body are considered to be oocytes in telophase. Alternatively, oocytes.which have one clear and distinct polar body with no evidence of protrusion are considered to be oocytes in metaphase. Methods of enucleating an oocyte, e.g., a caprine oocyte, are described in further detail in the Examples.
Irradiation The oocyte can be functionally enucleated by inactivating the endogenous DNA of the oocyte,using irradiation. Methods of using irradiation are known in 50409-13D(S) the art and described, for example, in Bradshaw et al. (1995) Molecul. Reprod.
. .
Dev. 41:503-512 .
Chemical lnactivation The oocyte can be functionally enucleated by chemically inactivating the endogenous DNA of the oocyte. . Methods of chemically inactivating the DNA
are known in the art. For example, chemical inactivation can be performed using the etopsoide-cycloheximide method as described in Fulkaj and Moore (1993) Molectel. Reprod Dev. 34:427-430 .
Introduction of a Functional Chromosomal Genome into an 0ocyte Methods described herein can include the introduction of a functional chromosomal genome into an oocyte, e.g., a functionally enucleated oocyte, e.g., an enucleafed oocyte, to form a reconstructed embryo. The functional chromosomal genome directs the development of a cloned or twansgenic animal which arises from the reconstructed embryo. Methods which result in the transfer of an essentially intact chromosomal genome'to the oocyte can be used.
Examples include fusion of a cell which contains the functional chromosomal genome with the oocyte and nuclear injection, i.e., direct transfer of the nucleus into the oocyte. .
Fusion Fusion of the somatic cell with an oocyte can be performed by; for example, electrofusion, viral fusion, biochemical reagent fusion (e.g., HA
protein), or chemical fusion (e.g., with polyethylene glycol (PEG) or ethanol).
Fusion of the somatic cell with the oocyte and activation can be performed simultaneously. For example, the nucleus of the somatic cell can be deposited within the zona pelliduca which contains the oocyte. The steps of fusing the nucleus with the oocyte and activation can then be performed 50409-13D(S) simultaneously by, for example, applying an, electric field. Methods of simultaneous fusion and activation of a somatic cell and an oocyte are described herein.
Activation of a Recombinant Embryo Activation refers to the beginning of embryonic development, e.g., replication and DNA synthesis. Activation can be induced by, for example, electric shock (e.g., in electrofusion), the use of ionophores, ethanol activation, or the oocyte can be obtained during a stage .in which it is naturally activated, e:g., an oocyte in telophase.
Electrofusion A reconstructed embryo can be activated using electric shock, i:e., electrofusion. The use of electrofusion allows for the fusion of the somatic cell - with the oocyte and activation to be performed simultaneously.
Chambers, such as the BTX 200 Embryomanipulation System, for carrying out electrofusion are commercially available from, for example, BTX, San Diego. Methods for performing electrofusion to fuse a somatic cell, e.g., a caprine somatic cell, and an oocyte, e.g., an enucleated oocyte, e.g., an enucleated caprine oocyte, are described herein:
Ionophores In addition, the reconstructed embryo can be activated by ionophore activation. Using an ionophore, e.g., a calcium.ionophore, the calcium . concentration across the membrane of the reconstructed embryo is changed. As the free calcium concentration in the cell increases, there is a decrease in phosphorylation of intracellular proteins and the oocyte is activated. Such methods of activation are described, for example, in U.S. Patent Number 5,496,720.

50409-13D(S) Ethanol Activation Prior to enucleation, an oocyte, e.g., an oocyte in metaphase II, can be activated with ethanol according to the ethanol activation treatment as described in Presicce and Yang (1994) Mol. Reprod. Dev. 37:61-68, and Bordignon and Smith (1998) Mol. Reprod. flev: 49:29-36 .
Ooctyes in Telophase Oocytes in telophase are generally already activated. Thus, these cells often naturally exhibit a decrease in calcium concentration which prevents fertilization and allows the embryo to develop.
Transfer of Reconstructed Embryos A reconstructed embryo of the invention can be transferred, e.g., implanted, to a recipient doe and allowed to develop into a cloned or transgenic mammal, e.g., a cloned or transgenic goat. For example, the reconstructed embryo can be transferred via the fimbria into the oviductal lumen of each recipient doe as described below in the Examples. hl addition, methods of transferring an embryo to a recipient mammal are known in the art and described, for example, in Ebert et al. (1994) BiolTechnology 12:699.
The reconstructed embryo can be maintained in a culture until at least first - cleavage (2-cell stage) up to blastocyst stage, preferably the embryos are transferred at 2-cell or 4 cell=stage. Various culture media for embryo development are known in the art. For example, the reconstructed embryo can be co-cultured with oviductal epithelial cell monolayer derived from the type of mammal to be provided by the.invention. Methods of obtaining goat oviductal epithelial cells (GOEC), maintaining the cells in a co-culture are described in the Examples below.
Purification of Proteins from Milk i~VO 00126357 PCTlLJS99/25710 The transgenic protein can be produced in milk at relatively high concentrations and in large volumes, providing continuous high level output of normally processed peptide that is easily harvested from a renewable resource.
There are several different methods known in the art for isolation of proteins form milk.
Milk proteins usually are isolated by a comuination of processes. ltaw ..
milk first is fractionated to remove fats, for example; by skimming, centrifugation, sedimentation (H.E. Swaisgood, Developments, irz Dairy Chemistry, I: Chemistry of Milk Protein, Applied Science Publishers, NY, 1982), t0 acid precipitation (LT.S. Patent No. 4,644,056) or enzymatic coagulation with rennin or chymotrypsin (Swaisgood, ibid.}. Next, the mayor milk proteins may be fractionated into either a clear solution or a bulk precipitate from which the specific protein of interest rgaay be readily purified:
French Patent No. 2487642 describes the isolaticin of milk proteins from skim milk or whey by membrane ultrafiltration in combination with exclusion chromatography or ion excharAge chromatography. ~Ilaey is first produced by removing the casein by coagulation with rennet or. lactic acid. U.S. Patent N~.
4,485,040 describes the isolation of an alpha-Iactogiobulin-enriched product in the retentate from whey by two sequential ultrafiltration steps. U.S. Patent No.
4,644,056 provides a rriefri~d for purifying irnmunogiobulin frorri riiiik ~r colostrum by acid precipitation at pI-I 4.0-5.5, and sequential cross-flow filtration first on a merribrane with 0.1 - 1.2 micrometer pare size to clarify the product pool and.then on a membrane with a separation limit-of 5 - 80 kd to concentrate lt.
Similarly, U.S. Patent No. 4,897,465 teaches the concentration and enrichment of a protein such as imrnunoglobulin from blood serum, egg yolks or whey by sequential ultrafiltration on metallic oxide membranes with a pI~
shift.
Filtration is carried out first at a pH below the isoelectric point (pI) of the selected protein to remove bulk contaminants from the protein retentate, and next at a pH
3o above the pI of the selected protein to retain impurities and pass the selected 50409-13D(S) protein to the permeate. A different filtration concentration method is taught by European Patent ~No. EP 467 482 B 1 in which defatted skim milk is reduced to pH 3-4, below the pI of the milk proteins, to solubilize bothcasein and whey proteins. -Three successive rounds of ultrafiltration or diafiltration then concentrate the proteins to form a retentate containing 15-20% solids of which 90% is protein. Altematively,.British Patent Application No. 2179947 discloses the isolation. of lactoferrin from whey by ultrafiltration to concentrate the sample, followed by weak cation exchange ,chromatography at approximately a neutral pH. No measure of purity is reported. In PCT Publication TTo. Wp 95!22258, a protein such as -lactoferrin is recovered from milk-that has been adjusted to high ionic strength by the addition of concentrated salt, followed by cation exchange chromatography.
In all of these methods, milk or a fraction thereof is first treated to remove fats, lipids, and other particulate matter that would foul filtration membranes or ~5 _ chromatography media. The initial fractions thus produced may consist of casein, whey, or total milk protein, from which the protein of interest is then isolated.
PCT Patent Publication No. W0. 94/19935 discloses a method of isolating a biologically active protein frorn'whole milk'by ,stabilizing the solubility of total milk proteins with a positively charged,agent such as aTginine, imidazole or 2o Bis-Tris. This treatment forms a.clarified solution from which the protein may be isolated, e.g., by filtration through membranes that otherwise would become clogged by precipitated proteins.
U.S. Patent No. 6,268,487 discloses a method for isolating a soluble milk component, such as a peptide, in its biologically active form from whole milk or a 25 milk fraction by tangential flow filtration. Unlike previous isolation methods, this eliminates the need for a first fractionation of whole milk to remove fat and casein micelles, thereby simplifying the process and- avoiding losses of recovery and bioacdvity. This method may be used in combination with additional purification steps to further remove contaminants and purify the product, e.g., 30 protein, of interest.

50409-13D(S) This invention is fu~rther.illustrated by the following examples~which in no way should be construed as being further limiting.
5. Examples Donors and recipients used in the following examples weredairy goats of .
the following breeds (mixed or not): Alpine, Saanen, and Toggenburg. All 'goats were maintained at the Genzyme Transgenics faun in Charlton, Massachusetts.
Collections and 'transfers were completed during the spring and early summer (off season). . . .
Isolation of Caprine Somatic Cells.
Caprine fetal fibroblast cell lines used as karyoplast donors were 'derived from six day 35-40 fetuses produced by artificially inseminating non-transgenic does with fresh collected semen from a tiansgenic antithrombin III (ATIII) founder buck. An ATIII cell line was chosen since it provides a well .
characterized genetic marker to the somatic cela lines, and it targets high, level expression of a complex glycosylated protein (ATIII) in the milk of lactating does. Three fetuses which were derived from the semen of the transgenic ATIII
. buck were surgically removed at day 40 post coitus and placed in equilibrated Ca'''/Mg"-free phosphate buffered saline (PBS): Cell suspensions were prepared by mincing and digesting fetal tissue in 0.025% trypsinl0.5 mM EDTA at 37°C
for ten minutes. Cells were washed with equilbrated Medium.199T""
(M199)(Gibco) + 10% Fetal Bovine Serurri (FBS) supplemented with nucleosides, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1%
penicillin/streptomycin (10,000 LU. each/ml) (fetal cell medium), and cultured in 25 cm~, flasks. The cultures were re-fed 24 hours later with equilibrated fetal cell WO OOI26357 PC'd7E3~991257~t9 medium. A confluent monolayer of primary fetal cells was harvested by.
trypsinization on day four by washing the monolayer twice, with ~a++~Mg~'+-free PBS, followed by incubation with 0.025% trypsin10.5 rnM EDfiA at 38°C for 7 m inutes.
Cells potentially expressing A°fIII were then prepared for cryopreservation, or maintained as stock cultures.
Sexing az~d Genoty,~aing oJ'I~ozz~r C'~11 Lanes Genomic DNA was isolated from fetal head tissue for ATIII donor 1,0 karyoplasts by digestion with proteinase h followed by precipitation with isopropanol as described in Laird et al. (1991) Nucleac acid Res. 19:4293, and analyzed by poIyrzierase chain reaction (PCI~) for the presence of human W ntithrombin Ill, (A T IiI) sequences as wail as for sexing. i ne A T iia sequence is part of the BC6 construct (Goat Beta-Casein - human ATIII cDNA) used to generate the ATIII transgenic line as described in Edmunds et al. (1998) Mood 91:4561-4571. The human ATIII sequencewas detected by amplification of a 367 by sequence with oligonucleotides GTC11.and GTC12 (see below). For sexing, the zfX/zfY primer pair was used (see below) giving rise to a _445.bp (zfX)/447 by (zfy) doublet. l3pon digestion with the restriction ei~zzyme Sacl (hIew England l3iolabs), the zfX band was cut into ~vo small fragments (272 and i 73 'np).
Males were identified.by the presence of the uncut.447 by zf~' band. .
For the PCR reactions9 approximately 250 ng of genomic DNA was diluted in 50 ml of.PClz buffer (20 mM Tris pII 8.3, 50 rnM KCl and 1.5 mM
MgClz, 0.25 mM deoxynucleotide triphosphates, and each primer at a concentration of 600 mM) with 2.5 units of Taq polymerise and processed using the following temperature program:
l cycle at 94°C 60 seconds 5 cycles at 94°G 30 seconds 58°C 45 seconds _.

'WO 00/2637 ~CTdUS93125710 74°C 45 seconds 30 cycles at 94°C 30 seconds SS°C 30 seconds 74°C 30 seconds The following primer set was used to detect the human A'I'III sequence:
GTC 11: CTCCATCAGTTGCTGGAGGGTGTC.ATTA (SEQ ID N~:1 GTC 12: GAAGGTTTATCTTTTGTCCTTGCTCaCTCA (SEQ ID N~:2) The following primer set was used for sexing:
~ zfX: ATAATCACATGGAGAGCCACAAGC (S:EQ ID NO:3) zfY: GCACTTCTTTGGTATCTGAGAAAG (SEQ.ID N0:4) Two of the fetuses were identified to be a~r~ale and were both negative for U $ne A T III sequence. An~ther fetliiS ~V'aS ~d~Ylt1 led 'atS fcat3aic and C:vu iaaaWd positive for the presence of the ATIII sequence.
Preparatioaa ofATIIl ~xpressir~g Do~aor Celds for Er~zbryo Reconstitution A transgenic female line (CFFI55-92-6) originating from a day 40 fetus 25 was identified by PCR analyses, as described above, and used for alI
nuclear transfer manipulations: Transgenic fetal fibroblast cells were maintained in cmz flasks with fetal cell medium, re-fed on day :four following each passage, and harvested by trypsinization on day seven. From each passage, a new 25 cm'-flasks was seeded to maintain the stock culture.- Briefly, fetal cells were seeded in 30 4-well plates with fetal cell medium and rnaintai:ned in culture (5% C~JZ
at 39°C).
_77_ WO 00/26357 PC'T/iJ~99/257t~
Forty-eight hours later, the ynedium was replaced with fresh fetal cell medium containing 0.5% FBS. The culture was re-fed every 48-72 hours over the next seven days with fresh fetal cell medium containing 0.5% FI3S. On the seventh day following first addition of fetal cell medium (0.5% F)3S), somatic cells used as karyoplast donors were harvested by trypsinization as previously described.
'The cells were resuspended in equilibrated M199+10% FI3S supplemented with 2mM L-glutamine, 1% penicillinlstreptomycin (10,000 LIJ. each/ml) one to three hours prior to fusion to the enucleated oocytes.
10' Krxryotypiaag of Cell Lifaes 'The clonal lines were fu~her evaluated by karyotyping to determine gross chromosomal abnormalities in the cell Iines. Cells were induced to an-est at metaphase by incubation daith 0.02 p.g/anl of I~emecolcine (Sigma) for 12 hours.
After trypsinization, the resulting pellet was suspended in a hypotonic solution of ~5 mM I~.Cl in water and incubated at 37°C for 20 minutes. Cells were fixed for 5 minutes each time in 3 changes of ice-cold acetic acid-methanol (I :3) solution before drops of the cell suspension were placed in pre-washed microscopic slides.
Following air-drying, chromosome preparations were stained with 3% Caiernsa stain (Sigma) in P13S for 10 minutes. The chromosome spreads were counted for a as a'annn_. r.......~:~w s:.. .1 '~' 2~ eatifl felt lifle at ivvv~i aiaa~aatiea;~savi3 ui'rW"ca via iaiaaufirjivia.
Immmaohist~chenaiccal ~4nafysis Antibodies specific for vimentin (Sigma) and pan-cytokeratin (Sigma) were used to characterize and confirm the morphology of the cell lines. Cells were plated in sterile gelatin coated cover slips to 75% confluency and fixed in 2% paraformaldehyde with O.OS% saponin for 1 hour. Cells were incubated in 0.5% PVP in P13S (PBS/PVP) with primary antibodies for 2 hours at 37 °C, rinsed with 3 changes of PBSII'~IP at 10 minute intervals, and incubated for 1 hour in secondary antibodies conjugated with Cy3 and FITC respectively.
Alkaline phosphatase (Sigma) activity of the cells was also performed to _72_ wo oons~s~ pcTms99nsm o determine the presence or absence'of undifferentiated cells, The cover slips were rinsed and subsequently mounted 'on glass slides with 50% glycerol inPBSlPVP
with 10 p,g/ml bisbenzimide (H-33342, Sigma) and observed under fluorescent rntcroscopy..
Epithelial anil fibroblast lines positive for vimentin and pan-cytokeratin, respectively, and negative for alkaline phosphatase activity were generated from .-the ATIII primary cultures. In the cell cultures, tvvo morphologically distinct cell types were observed. Larger."fibroblast-like" cells stained positive for virnentin .
and smaller "epithelial-like" cells stained positive for pan-cytokeratin which 0 coexisted in the prirxaary cell cultures. The isolated fibroblast lines from ATIII
shoved a tendency to differentiate into epithelial-like cells when cultured for 3 days after reaching confluency. Subsequent passages induced selection against fibroblast cells giving rise to pure epithelial cells ;as confirmed by the lack of positive staining for vimentin. Senesces or possible cell cycle arrest was first obseived at passage 28. These cells appear bigger in size, (>30 pm) compared to the normally growing cells (15-25.um) and can be maintained in culture in the absence of apparent mitotic activity for several months without loss of viability.
Embryo reconstruction using nuclei from the arrested cells produced morula stage embyos suggesting reacquistion of mitotic activity.

Donor Karyoplast Cedl Cycle Synchronization and Clzaracterization Selected diploid transgenie female cell lines were propagated, passaged sequencialiy and cyrobanked as future karyoplast stock. Donor karyopla~sts for nuclear transfer were seeded 'in 4 well plates arid cultured for up to 48 hours in DMEM + 10% FBS or when cells reached 70-80% confluency. Subsequently, the cells were induced to exit growth phase and enter the quiescent. stage (G°) by serum deprivation for seven days using DMEM supplemented with 0.5% FBS to synchronize the cells.. Following synchronization at G°, a group of cells were induced to re-enter the cell cycle by resuspending; the cells in M199 + 10%
FBS
up to thxee hours prior to karyoplast-cytoplast fusi~n to synchronize the cells at WO OOI263S7 PC°rltJ~991257t0 the early G, phase prior to STAI~'I°. , A second group of cells were also released from the quiescent state and cultured in Ie~Il99 + 10% FBS for l2 or 36 hours to synchronize cells at the S-phase. Cells v~ere harvested by standard trypsinisation and resuspended in 1VI199 -+- 10% FBS and electofused as lcaryoplasts donors within I hour 'The metaphase spreads from the transgeriic cell lines carrying the ATIII
construct at passage 5 was 81 % diploid and this did not alter significantly at passage 15 where 78% of the spreads were diploid.
Ceti cycle synchrony eves determined by immunohistoehemical analysis using antibodies against cyclin ~l, 2, 3 and PCNA (~ncogene Research Products) fox the absence of the protein complex to indicate quiescence (G°) or presence of the complex to indicate G, entry. Cells in the presumed S-phase of the cell cycle were identified by the presence of DNA synthetic activity using the thymidine analog 5-bromo 2'-deoxyuridine-5'triphospate (BrDu, Sigzria) and streptavidiil-Biotin l3rDu stai~~ing 1<it (Gncogene Igesearch Products):
Immunofluorescence analysis of cells subjected to the synchronization regimen demonstarted that following seven days of serurr~ deprivation, 90°!~ of the cells were negative for Ci, stage cyclins D 1, 2"3 and PNCA, and were therefore in G° arrest. Restoration of the serum content to 10% for this line induced reentry i~ato the cell cycle with approximately 74% of the calls reaching early G; within 3 hours following serum, addition based on positive staining for cyclins D l, 2, 3 and PCNA. Saturn restoration for 12 to 36 hours showed that 89% of the cells were positive far BrLW indicating DNA synthetic activity. lr~
this study, clonal lines generally responded differently to the serum synchronization regimen. , An indirect relationship was observed where the rate of cell synchronization decreases with the increase in passage numbers. Further, as passage number increased the population doubling times decreased, each clonal cell line revealed a decreased sensitivity to serum synchronization of the cell cycle.
_74.

~V~ 00/26357 PCTltIS99125710 Superovulation of Doraor Goats arid Gocyte Collection Estrus was synchronized on day ~ by a 6 mg subcutaneous Norgestomet ear implant (Synchro-mate B). A single injection of prostaglandin - (PGF2a.)(Upjohn US) was administered on day 7: Starting on day 12, FSI-I
(Folltropin-V, Vetrepharm, St Lauremt, Quebec, Canada) was administered twice daily over four consecutive days. The ear implant was removed~n day 14. -Twenty-four hours following implant removal, the doinor animals were mated several times to vasectornized rizales over a 48 hour interval. A single injection of GnRH (Rhone-Merieux US) was administered intramuscularly following the last FSH injection. Oocytes were recovered surgically from donor animals by mid-ventral laparotomy approximately 18 to 24 hours following the last mating" by flushing the oviduct with Ca~"IMg*~ -free PBS prewarmed at 37°C.
Oocytes were then recovered and cultured in equilibrated M 199+10%FBS supplemented with 2mM L-glutarnine, 1% penicilliii/streptomycin (10,000 LU. each/ml):
~ocyte Enucleatiorr do vivo matured oocytes were collected from donor goats. Oocytes with attached cumulus cells or devoid of polar bodies were discarded. Cumulus-free oocytes were divided into two groupsa oocytes with only oiie polar body evident (metaphase II stake) and the activated telophase II protoc~1 (oocytes with one polar body and evidence of an extruding second polar body). Oocytes in telophase II were cultured iii M199 +.IO% FBS for 2 to 4 hours. Oocytes that had activated during this period, as evidenced by a first polar body and a partially extruded second polar body9 were grouped as culti.ire induced, calcium activated telophase II oocytes (Telophase II-Ca2+) and enucleated. Oocytes that lead not activated were incubated for 5 minutes in PBS containing 7% ethanol prior to enucleation. Metaphase II stage oocytes (one polar body) were enucleated with a 25-30 micron glass pipette by aspirating the first polar body and adjacent _ cytoplasm surrounding the polar body (approximately 30% of the cytoplasm) . presumably containing metaphase plate.

W ~. 00!26357 ~CTIdJS991257 t t6 As discussed above, telophase stage o~cytes were prepared by two procedures. Oocytes were intiaIIy incubated in phosphate buffered saline (PBS, Caz+lMg2+ free) supplemented with 5% ~I3S for'15 minutes and cultured in M199 + 10% FBS at 3S°C for approximately three hours until the telophase spindle configuration or the extrusion of the second polar body was reached. All the oocytes that responded to the sequential culture under differential extracellular -calcium concentration treatment were seperated and grouped as Telophase lI-Ca2~.
The other oocytes that did riot respond were further incubated in 7% ethanol in M199 + 10% FBS for 5-7 minutes (Telophase Il-ETOH) and cultured in M199 +
10% CBS at 38°C for another 3 hours,until.the telophase ll spindle configuration was reached. Thereafter, the oocytes were incubated in 30-50 ~tl drops of M199 +
10% FBS conatining 5 ~.gli l of cytochalasin-B for 10-15 minutes at 38°C.
Oocytes were enucleated 'uitle a 30 micron, {OD) glass pipette by aspirating the first polar body and approximately 30% of the adjacent cytoplasm containg the metaphase ii or anout 10% of the cytoplasm containing the telophase di spir~die.
After enucleation the oocytes were immediately reconstructed.
EntbYyo Recorc~truction CFF155-92-6 somatic cells used as karyoplast donors were harvested on 2n clay 7 iJy tr~pci;ri~i_n-g ~f~,~2~°/n fryr~ci-_n-/Q:~ m_,M_ F~T_A_)f~igvn~;) f9_r ? 7tl~inut~S-Single cells were resuspended in equilibrated M199+10% FBS supplemented with 2mM L-glutamine, penicillin/streptomycin. The donor cell injection was carried out in the same medium as for enucleation. Donor cells were graded into small, medium and large before selection for injection to enucleated cytoplasts.
Small single cells {10-15 ynicron) were selected with a 20-30 micron diarrieter glass pipette. The pipette was introduced through the same slit of the zone made during enucleation and donor cells were injected between the zone pellucida and the ooplasmic membrane. The reconstructed embryos were incubated in M199 30-60 minutes before fusion and activation.
Jl7 -7f-W~ 00/26357 t'C'f/tJS99/25710 Fusion and Activation All reconstz-ucted embryos (ethanol pretreatment or not) were washed in fusion buffer {0.3 M mannitol, 0.05 mM CaCl2, 0.1 a~rzM MgS04, 1 mM K~I-IPOa, Q.l mM glutathione, 0.1 mglznl BSA in distilled water) for 2 minutes before electrofusion. Fusion and activation were carried out at room temperatuz~e, in a chamber with two stainless steel electrodes 200 microns apart (BTX 200 ..
Embryomanipulation System, BTX-Genetrorzics, San Diego, CA) filled with fusion buffer. Reconstructed embryos were placed with a pipette in groups of 3-and manually aligned so the cytoplasrr~ic membrane of the recipient oocytes and donor CFFI55-92-6 cells were parallel. to the electrodes. Cell fusion and activation were simultaneously induced 32-42 hours post GnRH injection with an initial alignmentllzolding pulse of S-l0 V AC for 7 seconds, followed by a fusion pulse of .1.4 to 1.8 KV/cm DC for 70 microseconds using an Electrocell Manipulator and Enhancer 400 {BT'X-Genetronics). Embryos were washed in fusion medium for 3 minutes, then they were transferred to M199 containizig 5 ;~g/rnl cytochalasin-B (Sigma) and 10% FBS and, incubated for 1 hour. Embryos .
were removed from M199/cytochalasin-B medium and cocultured in 50 microliter drops of M199 plus 10% FBS with goat oviductal epithelial cells overlaid with paraffin oil. Embryo cultures were maintained in a humidified 39°C incubator with 5% CO, for 48 hours before transfer ~f the. embryos to recipient does.
Reconstructed embryos at 1 hour following; simultaneous activation and fusion with G°,G, and S-phase karyoplasts all showed nuclear envelope breakdown (NEBD) and premature chromosome c;ondensation.(PCC) when the cytoplasts were at the arrested metaphase II stage. Subsequent nuclear envelope formation was observed to be at about 35% at 4 he~ur post activation. Oocytes reconstructed at telophase.Il stage showed that an average of 22% of oocytes observed at 1 hour post fusion of G°,G, and S-phase karyoplast underwent NEBD
and PCC, whereas the remaining oocytes have intact nuclear lamina surrounding the decondensing nucleus. No consistent nuclear morphology.other than lack of, WO 00126357 PC'r/US99/257t0 of the occurrence of NEBD and PCC was observed between the metaphase and two telophase reconstruction prot~cols employed. Differences became evident when cloned embryos were observed to have a higher incidence of advanced cleavage stages (8 to 32 .blastomeres~ when embryos were reconstructed with S-phase donor nuclei cozripared to when Gaox G, stage karyoplasts were used (2 to 8 blastomeres) following culture t~8 vitYO for 36 -to 48 hours. Fluorescent _.
microscopy analysis showed that the.nuclei of some of the rapidly dividing embryos were fragmented. Other embryos developed to the 32 to 64 cell stage within 3 days of culture before cleavage development was,blocked. Analysis of blastomere and nuclei numbers of these embryos showed the failure of synchronous occurrence of cytokines and karyokinesis wherein blastomeres were either .devoid or their corresponding nuclei or contained multiple nuclei. In contrast, morphologically normal looking embryos showed synchronous cytokinesis and karyokinesis.
Goat Oviductctl ~patheliczl Cells (Ci~EC)lReconstructed Enabr~o Coculture G~EC were derived from oviducfal tissue collected during surgical ~viductal flushing performed on synchronized and superovulated does. ~viductal . tissue from a single doe was transferred to a sterile 1 S ml polypropylene culture ~.. -roa,.-.,ø ,~ ~q~ ~ c~n a of as~, ~ ~~ t "a~ 'n o illbe li~~lta~rlllilg J 1111 Vd Ciai~,i111ilipl'S.°u l~t~~, a0/u Ft7J, ~. 'ana,'a i..°glusumi v, penicillin/strepomycin. A single cell suspension was prepared by irottexing for l minute, followed by culture in a humidified S% C02 incubator at 38°C
for up to one hour. . The tube was vortexed a second time for one minute, then cultured an additional five minutes to allow debris to settle. The top four millimeters containing presumed single cells was Transferred to a new I S ml culture tube and centrifuged at 600x g for 7 minutes, at room temperature. The supernatant was removed, and the cell pellet resuspended in 8 ml of equilibrated GOEC medium.
The GOEC were cultured in a 2S cmz flask, re-fed on day 3, and harvested by trypsinization oxa day six, as previously described. Monolayers were prepared weekly, from primary (p~1JC cultures, for each experiment. Cells were _70_ CVO 00/26357 PC'~I17S99125710 resuspended in GOEC medium at Sx1051m1, and 50 microliter/well was seeded in 4-well plates (lSmm). .The medium was overlaid with 0.5 ml light paraffin oil, and the plates were cultured in a humidified 5% C~~'' incubator at 38°C. The cultures were re-fed on day two with 80% fresh equilibrated culture medium.
All reconstructed embryos were cocultured with the GUEC rrionolayers in vitro in incubator at 39°C, 5% C02 before transfer to recipients of GTC farm. ..
All experimental replicates for ATIII yielded cleavage stage embryos that were transferable on day 2 into synchronized recipients. Embryos using fibroblasts arid epithelial cell phenotype as donor karyoplasts showed cleavage and development in culture. The percentage of cleavage development was higher in reconstructed couplets that used preactivated telophase II stage cytoplasts (45%) and telophase II-ethanol activated (56.%) wren compared to cytoplasts used at metaphase II arrested (35%) using ATIII karyoplasts. There were no differences observed in the cleavage rates of embryos that were reconstructed using donor karyoplasts in G°, G, or S-phase of the cell cycle although, the morphological quality of embryos vVas better wherd doaaor karyoplasts were in as G° ~r.G, compared to S-phase. -Embryos were generally between the 2 to 8 cell stage with the majority of the embryos having 3=4 blastorueres at the time of transfer. Normal cleavage development corresponded chronologically to approximately 36 to 48 hours post fusion and activation. IvIorphoiogicaiiy normal appearing embryos were selected at the 2 to 8 cell stage following development in vitro for 36 to 48 hors.
Estrus Synchrortizatzon of Recipient does Hormonal treatments were delayed by 1 day for recipients (as compared to donors) to insure donor/recipient synchrony. Estrus was synchronized on day 1 by a 6 mg subcutaneous norgestomet ear implant. A single injection of prostaglandin was administered on day 8. Starting on day 14, a single intramuscular treatment of PMSG (CalBiochem IJS) was administered. The ear 3o implant was removed on day 15. Twenty-four hours following implant removal, WO 00126357 t'C'&'ltJ~99/257t0 recipient does were mated several times to vasectomized males over three consecutive days.
Enzbryo Transfer to Recipienat does Reconstructed embryos were co-cultured with G~EC monolayers for approximately 4~ hours prior to transfer to synchronized recipients.
Immediately -prior to transfer, reconstructed embryos were placed in equilibrated Ham's F-medium + 10% F13S. 'Two to four reconstructed embryos were transferred via the fimbria into the oviductal lumen of each recipient. Transfers were perfotTned in a minimal volume of IIIams's F-12 medium -~- 10% FBS using a sterile fire-polished glass micropipet.
The development of embryos reconstructed by nuclear transfer using transgenic caprine fetal fibroblasts and i~a vivo derived oocytes is summarized in Table 1. There was a, total of 14 .rounds of collection and transfers, with 4 donors set up for cGllection and ~--5 recipient does set up for transfer 4~ hours later. The three different enucleati~n/activation protocols were employed: Metaphase II, Telophase, and l~Ietaphas~ II pretreated with Ethanol: Following fission-activation, reconstructed embryos were co-cultured with primary goat epithelial cells; at least until cleavage (2-cell stage) up to early 1 fs-cell stage;
with most embryos being transferred at chronologically correct,2- and 4-cell stages. X11 transfers were surgical and oviductal, in hormonally synchronized recipients (due to the season). Rates of development were slightly superior when using the Telophase protocol and Ethanol protocol as compared to the Metaphase II
protocol. This is partly due to the fact that enucleation of the second polar body seems less traumatic for the oocytes, and partly due to what seems to be higher.
activation rate for oocytes pretreated with ethanol.
Table 1: Development of caprine embryos reconstructed by nuclear transfer of transgenic fetal fibroblasts. Three enucleation/procedure were used: Metaphase II
{first polar body enucleation), Telophase (second polar body enucleation), VVO 00/2b357 P~T/US99I25710 Ethanol (preactivation of Metaphase II stage oocytes by ?% ethanol treatment prior to enucleation). In all cases, concomitant fusion and activation was used.
Enucleation oocytes Oocytes Embryos Embryos And Reconstructedlysed Cleaved ~'ransferred -activation ( o ) ( o ) , protocol Metaphase II 138 67(48.5) 48(35) 47 .

Telophase 92 38(41) 41(44).. 38 Ethanol 55 23(42) 31(56) 27 FoIiowing embryo transfer, recipient does were examined by ultrasound, as early as day 25. ~-Iigh pregnancy rates ranging from 55-78% for ATIII
recipient does were diagnosed. For all three enucleation/activation protocols, it was observed that high proportion of does (65%) appeared. positive at day ~0.
However, it must be noted that, in most cases, fetal ;heartbeats could not be detected at such an early stage. Moreever, the positive ultrasound signal detected at day 30 was not characteristic of normal embryo development and appeared closer to vesicular development not associated with the formation of an embryo proper. This kind of embryonic development is not typically observed in other caprine embryo transfer programs (for example with microinjected embryos).
Biweekly, examination of these vesicular developments between day 25 and day ,,n w.r_.,v...a 6z.,.:. +z .~ 1. i .,.~ ~t da~r 4C) mr~o"ct ref the ~tV cW auai5ucu mame:jc prcg ",aaaavi28 w2rc. ava.~.~:~.il. aaau J , ...
fetuses were reabsorbed and normal ultrasound images were not apparent.
However, far 2 pregaiancies, heartbeats were detected by day 40. In these 2 cases, ultrasound examination between day 25 and day 40, not only detected a heartbeat, but also showed the development of recognizable embryonic strictures.
One of these pregnancies was established using the; Metaphase II
enucIeation/activation protocol, fusing the enucleated cytoplast to a quiescent karyoplast originating from a passage 6 culture of the CFF I55-92-6 fibroblast cell line. In this instance, 4 four-cell stage reconstructed embryos were transferred to the oviduct of the recipient doe. The other pregnalzcy {twins) was obtained from embryos reconstnacted according to the Tel~phase enucleation/activation WO 00/26357 ~'C'1'/US99/25790 protocol, fusing an ertucleated cytoplast derived from preactivated telophase Caz+
oocytes and G, karyoplasts originating from a passage 5 culture of the CFF I
55- r 92-6 epithelial cell line. In this case, 3 reconstructed embryos (1 two-cell stage and 2 four-cell stage) were transferred to the oviduct of the,recipient doe.
No pregnancies were observed with embryos generated by the Ethanol enucleation/activation protocol. I-lowever, numbers are i~ot large enough to conclude on the relative efficacy of the 3 enucleation/activatiom protocols used in this study.
Table 2: Induction of pregna~a~y and further development following transfer ~f eaprine embryos reconstructed with transgenic fetal fibroblasts and activated according to three protocols Enucleation RecipientsUltrasound Results . Term --~
~

Activation (average (positive/total recip) prec~nancie #

protocol of embryos/

recip) 30 days 40days 50 days Metaphase J

II 15(3.1) 9/i5 1/15 2/15 1 Telophase 14(2.7) 11/14 1/14 1/14 1 (twins) Ethanol 9(3) 5/9 0/9 0/9 0 Peri~aratcal Care of Recipieyat E~aabryos Does were monitored daily throughout pregnancy for outward signs of health (e.g.; appetite; alertness, appearance). Pregnancy was determined by ultrasonograph 25-28 days after the first day of standing estrus. Does were ultrasounded biweekly till approximately day 75 and there after once a month to monitor and assess fetal viability. Additionally, recipient does had serum samples drawn at approximately day 21 post standing estrus for serugn progesterone analysis. This was to determine if a functioning corpus luteum was present. and h~w this compared to the animal's reproductive status (i.e., pregnancy)., At approximately day 130, the pregnant does were vaccinated with tetanus toxoid and Closta-idium C8il~. Selenium ~ vitamin E (Bo-Se) and vitamins A, D, and 13 complex were given intramuscularly or subcutaneously and _82_ iv0 00/26357 P~TI~.JS99/25710 a dewormer was administered. T'he does were moved to a clean kidding stall on approximately Day 143 and allowed to acclimate to this new environment prior to kidding. Observations of the pregnant does were increased to monitor for signs of pending parturition. After the beginning of regular contractions, the does remained under periodic observation until birth occurred. If labor was not progressive after approximately 1 S minutes of strong contractions the. fetal ..
position was assessed by vaginal palpation. If the position appeared normal then the labor seas allowed to proceed for an additional 'i-30 minutes (depending on the doe) before initiating an assisted vaginal birth. If indicated a cesarean section 1 o was performed. When indicated, parturition was induced with approximately S-mg of PGF2oc (e.g. Lutalyse). This induction can occur approximately between I4S-155 days of gestation. Parturition generally occurs between 30 and 40 hours after the first injection. The monitoring process is the same as described above.
Once a kid was born, tlae animal was quickly towel dried and checked for gross abnormalities and normal breathing. Kids were immediately reraaoved from the dam. Once the animal was determined to be in good health, the umbilicus was dipped in 7% tincture of iodine. Within the first hour of birth, the kids received their first feeding of heat-treated colostrurn. At the time of birth, kids 2o received injections of selenium t~ vitamin F (uso-Se) and vitamins h, i.r, and is complex to boost performance and health.
The first transgenic female goat offspring vvas produced by nuclear transfer was bom after 154 days of gestation following the induction of parturition and cesarean delivery. The birth weight of the offspring was x.35 kg which is within the medium weight range of the alpine breed. The female twins were born naturally with minimal assistance a month later with a gestation length of 1 S 1 days. The birth weights of the twins were both 3.5 kg which are also within the medium weight range for twins of this breed. All three kids appeared normal and healthy and were phenotypically similar for coat color and expressing markings typical of the alpine breed. In addition, ;all three offspring were similar _83_ w~ 00126357 t'C'I'IL)S99/~57t0 in appearance to the transgenic founder buck. No distinguishable phenotypic influence from the breed of the donor oocyte (Saanen, Toggenburg lbreed) or the .
heterogeneous expression of the fetal genotype was observed.
Transgenfc CI~ned Goats In order to confirm that the three kids were transgenic for the BC6 construct comprising the goat beta casein promoter and the human A.TIII gene sequence, PCR amplification and southern analysis bf the segment of the transgene were perfosrned.
Shortly after birth, blood samples and ear skin biopsies were obtained form the cloned female goats and the surrogate darns. The samples were subjected to genomic I7I~TA isolation. Laird et al. (1991 ) Nucleic Acids Res.
19:4293. Each sample was first analyzed by PGR using AT HI specific primers, and then subjected to Southern bI~t analyses usrng the AT IH cI~RTA (Edmunds et 95 al. (i998) Blood 9i:45b1-4571). F'or each sample, 5 pg of genomie I~hIA was digested with EcoRI (IoTew England Riolabs, I3everly, NIA), electrophoresed i~
0.7% agar~se gels (SeaI~ern~, ft~E) and irnfnobilized on nylon membranes (MagnaGraph, MSI, ~Iestboro, MA) by capillary transfer following standard procedures. Laird et al. (1991) Nucleic Acids Res. 19:4293. Membranes were 2p prQl~ed with the 1.5 kb ~'h'h~ I ~o Sc~l I AT III cDIvIA fragment labeled with a, -3zP
dCTP using the Prime-It~ kgt (Stratagene, La Jolla, CA). I-Iybridization was executed at 65°C overnight. Church et al. (1984) Prot. Nail Acad: Sci.
~1S'.~.
81:199.1-1995. The blot was washed with 0.2 X SSC, 0.1 % ST)S and exposed to X-OMATT'" AR film for 48 hours.
PCR analysis confirmed that all of the kids were transgenic for the l3Cb construct comprising the g~at beta casein promoter and the human ATHI gene a sequence. Southern blot analysis demonstrated the integrity of the ~C6 transgene. Hybridization to a diagnostic 4.1 kb EcolZf fragment was detected for .
all three cloned animals, the cell lines and a transgenic posit':ve control, blrt not w~ 00126357 1'CT3'g1S99125710 for the two recipient does. As expected, due to cross hybridization of the ATIII
cDNA probe to the endogenous goat AT locus; a 14 kb band was detected in all samples.
In addition, fluorescence in situ hybridization (FISH) was performed to determine the integration site of the BC6 construct. . For typing of the cloned goats, whole blood was cultured for lymphocytes harvest. Ponce de Leon et al.
{1992} J. Hared. 83:36-42. Fibroblast cells and.lymphocytes were pretreated and hybridized as previously described in van de Corput et al. (1998) Histochem Ccll Biol. 110:432-437, and Klinger et al. {1992).,Qm. J. Ha~rracara. Genet. 51:55-65. A
digoxygen labeled probe containing the entare 14.7 kb BC6 transgene was used in this procedure. The TSA T~'-Direct system (NEN T"" L ife Science Products, Boston, MA} was used to amplify the signal. R-bands were visualized using DAPI counterstain and identified as in Di Berardino et al. (1987) J. ~lered.
78:225-230. A Zeiss Axioskop microscope mounted with a FIamamatsu Digital Camera was used with Image-Pro C~ Plus software {Media Cybernetics, Silver Spring, MD) to capture and process images.FISI-i analysis of blood cultures from each transgenic kid with probes for the BC6 transgene showed that all three carry a chromosome 5 transgene integration identical to thai: found in the metaphase 2o plates derived from the CFF6 cell line. Moreover; analysis of at least 75 metaphase plates for each cloned offspring confirmed that they are not mosaic far the chromosome 5 transgenec integrateon.
As final confirmation that all three kids are derived from the transgenic CFF6 cell line, PCR-RFLP analysis for the very polyrnorphic MI-iC class II

gene was undertaken. Typing for the second axon of tt~e caprine MHC class II
DRB gene was perfom~ed using PCR-RFLP Typing as described Amills et al.
(1996) Irramunopathol: 55:255-260. Fifteen microlitens of nested PCR product was digested with 20 units of Rsal (New England Biolabs, Beverly, MA}.
Following digestion, restriction fragments were separated at room teri9perature in gyp ~0/z635°7 ~~~'~~~9izs r~~
a 4 to 20 % nondenaturing polycrylalnide gel (1~VPT~" precast gel, Stratagene, La Jolla, CA) in the presence of ethidiuln bromide.
As illustrated by the Rsral digests of the I?RB gene second axon, the three cloned offspring are identical to each other and identical to the CFF6 donor cell line; whereas the recipient does carry different alleles.
,Induction o, f Lactation and T~cansgene Expression of Proteins zn Milk In order to determine whether the targeted mammary gland specific expression of human ATIII proteins were present in milk, the cloned transgenic prepubertal clones were transiently induced to lactate. . .At two rilonths of age, the cloned offspring was subjected to~a tyro week hormonal lactati~il-induction protocol. Horanonal induction of.lactation for the CFF6-1 female was performed as described in Ryot et al. ( 1989) Indian ~ Anima Res. 10:49-51. 1 he.~Fl~6-1 kid was hand-milked oncd daily to collect milk samples for AT III expression 95 analyses. All protein analysis methods 4.,rre described in lrdanunds et al.
(1998) Blood 91:4561-4571. Concentration of recombinant A'TIII in the milk was determined by a rapid reversephase HiPLC method using a Hewlett Packard 100 f3PLC (Wilmington, TAE) with detection at 214 nm. The ATIII activity was evaluated by measuring thrombin inhibition with a tw~-stage colorimetric 20 endpoint assay. ~Iestern blot analysis was performed ~itl~ an affinity purified sheep anti-ATIiI 1~RP c~njugated polyclonal antibody (Sero'Tec, ~xford, LJI~).
Samples were boiled for 30 seconds, in redueing sample buffer prior to loading onto a 10-20 % gradient gel (~wl Scientific). Electrophoresis was operated at 164 volts (constant) until the dye front ran off the gel.
25 At the end of the treatment, small milk samples of 0.5 to 10 ml were collected daily for 20 days. 'fhhe small initial volumes of milk, 0.5 to 1 lnl, were typical of the amounts seed in prepubertal female goats hormonally induced to lactate. The volumes increased to 1.0 ml per day by the time the female was dried off, 25 days after the onset. The concentration and activity of ATI3I in several of 30 the samples was evaluated. As pgeviously noted with does frown tills specific EC6 ~gg-WO 00!26357 P~T/1JS99125710 transgenic cell line, high levels of the recombinant ATIII was detected by , Western blot analysis. Edmunds et al. (1998) Blood 91:4561-4571. The concentration of recombinant ATIII in the milk of the cloned offspring was 5.8 grarris per liter (20.SU/ml) at day 5, and 3.7 grams per liter (14.6 U/ml) by day 9.
These were in Line with levels recorded during the early part of a first natural lactation of does from this 13C6 line (3.7 to 4.0 grams per liter).
1 o . Discussion:
Healthy transgenic goats were obtained by nuchar transfer of somatic cells to oocytes that were enucieated either in the arrested Metaphase II or the activated Telophase II-stage. These studies show that serum-starved cells used to generate term pregnancies are likely at the G~/G, transitaon following restoration with 10% serum.
Imrrunoflcresence screening revealed that after 7 days of serum starvation, fetal somatic cells were negative for G, stage cyclins D.1, D2, D3 and PCNA; whereas within 3 hours of 10% FBS serum-activation a majority (e.g.
approximately 70%) expressed these markers.
Deconstruction of an enucleated metaphase II arrested oocyte with the transfer of a nucleus from a don~r karyoplast synchronized at G° or G, of the,cell cycle following simultaneous fusion and activation mimic the chronological events 'occurring during fertilization. The successful dLevelopment to term and birth of a normal and healthy .tiansgenic offspring following the simultaneoais fusion and activation protocol is in contrast with procedures.employed in other studies that report the requirement for prolonged exposure of donor nuclei to elevated cytoplasmic MPF activity to support cha~omavdn remodeling and reprogramming. See Campbell et a1. (1996) Nature 380:64-66; V~ilmut et aI.
(1997) Nature 385:810-813; Schnieke et al. (1997) Science 278:2130-2133;
Cibelli et al, (1998) Science 280:I2S6-1258. This result challenges the WO 00/26357 PC'tYtJS99125710 contention that prolonged rera~odeling of the somatic nuclei in conditions of elevated MPF activity prior to activation is important for embryonic and fetal development to term. The results also demonstrate that a reconstructed embryo may not have a requirement for prolonged exposure of the donor nucleus to MPF
nor are NEBD and PCC entirely requisite events. Rather chromatin remodeling events involving NE13D and PCC are Iikely permissive effects of MPF activity w and, as such, may not be required for the acquisition of developmental competence or totipotency. Instead, these events are likely to serve to facilitate the acquisition of synchronicity between the cytoplast and the karyoplast.
These 10, events may even be detrimental if normal diploidy is not maintained when the donor nuclei are induced to undergo PCC with resultant chromosome dispersion due to an aberrant spindle apparatus due in part to MPF activity. Therefore, karyoplast and cytoplast synchronization with respect to cell cycle is important, first for maintenance of normal ploidy and, second far the proper induction of genome reactivation and subseguent acquisition of,developr~zental competence of reconstructed embryos.
Further support is pr~vided in the second method where chromatin-intact metaphase II arrested oocytes were activated to reduce MPF activity and induce the oocyte to exit the M-phase and enter the first mitotic cleavage.
~(~ flppr07C~~n$tCly J JlOUr~ pUSya~tIV'c~LF(Drll, illG oo(:y6eS S~VGIe ell~.ILlG4le(1 dt l~I(3~J lilac stage prior to the onset of Ci, and fused and simultaneously activated with a don~r karyoplast in G, prior to S'f'I' of the cycle. In addition, the simultaneous activation and fusion insured that tendencies of non-aged oocytes to revert back to an arrested state were circumvented. ilsing this paradigm, a noranal and healthy set of twin cloned transgenic kids were produced. This procedure inherently provides a homogenous synchronization regimen: for the cytoplast to coincide closer with the donor nuclei in G, prior to S'I°T. Further preactivatior~
of the oocyte induces a decline in cytoplasmic MPF activity, thus inhibiting the occurrence of NEBD and PCC. These results suggest that NEBD and PCC is only facultative for the induction of cytoplast and karyoplast synchr~ny but not m30_ necessary for acquisition of proper genome reactivation and subsequent . development to term of the nuclear transfer embryo using somatic cell nuclei.
These, findings further suggest that differentiated cells at the Go or G, stage function similar to embryonic blastomeres with respect to their ability to acquire totipotency when used in combination with an arrested or an activated recipient cytoplast. The use of both metaphase II arrested and telophase II cytoplasts provides dual options for cytoplast preparation in addition to providing an .
opportunity for a longer time frame to prepare the cytoplast. T'he use of Telophase II cytoplasts may have several practical and biological advantages.
The telophase appr~ach facilitates efficient enucleation avoiding the necessity for chromatin staining and ultraviolet localization. Moreover, enucleati~n at telophase enables removal of minimal cytoplasmic material and selection of a synchronous group of activated donor cytoplasts. This procedure, also allows for the preparation of highly homogenous group of donor nuclei to be synchronized ~ 5 with the cell cycle of the cytoplast. When used for embryo reconstruction, these populations showed a higher rate, of embryonic development in witr~o. 'f hus, reconstructed embryos comprised ~f a synchronously activated cytoplast and karyoplast are developmentally competent.
20 In addition to a successful transgenic founder production, nuclear transfer of somatic cells allows for the selection of the appropriate transgenic cell line before the generation of cloned transgenic embryos.- 'I°his is particularly important in the cases where several proteins are to be co-expressed by the transgenic mammary.gland. For example, in the transgenic production of 25 recombinant monoclonal antibodies in milk, heavy chain and light chain transgenes ideally should be expressed in the same secretory cells of the mammary epithelium at equivalent levels for the efficient production of intact . antibodies. In addition, transgenes expressing each protein should be co=
integrated in the same locus to favor equivalent expression and avoid segregation 30 of heavy chain and light chain transgenes during herd propagation..

50409-13D (S) The generation of transgenic'animals that have corripletely identical genetic backgrounds also enhances the possibility of studying the expression and secretion characteristics of recombinant proteins by the mammary gland.. For example, the availability~of several ~completely.identical trarisgenic females producing recombinant human ATIII will help determine the extent of variation i-n the carbohydrate structure of this.profein, as it is produced by the mammary , gland.. Thus, it may be feasible to improve the, characteristics of the recombinant proteins produces iri the transgenic animal system by varying environmental .
factors, (e.g., nutrition) or to increase the milk volume yield of lactation-induction protocols.to diminish further the time necessary to obtain adequate amounts of recombinant protein for pre-clinical or clinical programs.
The high-level expression of recombinant human ATIII detected in the.
~ 5 milk of the CFF6-1 cloned goat illustrates one of the most important aspects of this technology. By combining nuclear transfer with lactation-induction in~
prepubertal goats; it may be possible to characterize transgenic animals and the proteins they secrete in 8 to 9 months from the time of cell line transfection of milk expression.. The amount of milk collected in an induced lactation. is not only 20 sufficient to evaluate the recombinant protein yield, but, when. mg per ml expression levels are obtained, is adequate for more qualitative analyses w , (glycosylation, preliminary pharmaco-kinetics, biological and pharmacological activities). The continued availability o.f the transfected donor cell line also insures that genetically identical animals can be quickly generated, to rapidly 25 supply therapeutic proteins (with predictable characteristics) for clinical trials.
Other embodiments are within the following claims: .

Claims (38)

CLAIMS:

1. ~A method of making a transgenic non-human mammal comprising:
(a) fusing a non-human mammalian somatic cell capable of expressing a transgenic protein with a functionally enucleated oocyte, said functionally enucleated oocyte being from the same species as the somatic cell and being in the metaphase II stage of meiotic division, and the nucleus from said somatic cell containing at least one recombinant nucleic acid sequence to obtain a reconstructed embryo;
(b) activating the reconstructed embryo from step (a);
(c) maintaining the activated reconstructed embryo from step (b) in culture until the embryo is in the 2- to 8-cell stage of embryogenesis;
(d) transferring the 2- to 8-cell stage reconstructed embryo from step (c) into a female non-human mammalian recipient;
(e) allowing the transferred reconstructed embryo from step (d) to develop into a mammal thereby providing the transgenic non-human mammal.

2. ~The method of claim 1, wherein the reconstructed embryo is in the 2-cell stage of embryogenesis when transferred to the recipient.

3. ~The method of claim 1, wherein the reconstructed embryo is in the 4-cell stage of embryogenesis when transferred to the recipient.~

4. ~The method of claim 1, wherein the reconstructed embryo is in the 8-cell stage of embryogenesis when transferred to the recipient.

5. ~The method of any one of claims 1 to 4 wherein said at least one recombinant nucleic acid sequence is a DNA
sequence encoding a desired gene that is actuated by a tissue specific promoter.

6. ~The method of claim 5, wherein said tissue-specific promoter is a promoter preferentially expressed in mammary gland epithelial cells.

7. ~The method of claim 6, wherein said promoter is selected from the group consisting of a .beta.-casein promoter, a .beta.-lactoglobin promoter, whey acid protein promoter and lactalbumin promoter.

8. ~The method of any one of claims 1 to 7, wherein said transgenic non-human mammal is a goat.

9. ~The method of any one of claims 1 to 8, wherein said at least one recombinant nucleic acid sequence encodes a polypeptide selected from the group consisting of an .alpha.-1 proteinase inhibitor, an alkaline phosphotase, an angiogenin, an extracellular superoxide dismutase, a fibrogen, a glucocerebrosidase, a glutamate decarboxylase, a human serum albumin, a myelin basic protein, a pro-insulin, a soluble CD4, a lactoferrin, a lactoglobulin, a lysozyme, a lacto-albumin, an erythropoietin, a tissue plasminogen activator, a human growth factor, an antithrombin III, an insulin, a prolactin, and an .alpha.-1-antitrypsin.

10. ~The method of any one of claims 1 to 9, wherein said somatic cell is selected from a group of cell types present in a non-human mammal consisting of:

a) fibroblasts b) cumulus cells c) neural cells d) mammary cells; and e) myocytes.

11. ~The method of claim 10, wherein the fibroblast is an embryonic fibroblast.

12. ~The method of any one of claims 1 to 11, wherein the somatic cell is in G1 stage.

13. ~The method of any one of claims 1 to 11, wherein the somatic cell is in G0 stage.

14. ~The method of any one of claims 1 to 13, wherein the nucleus of the somatic cell is introduced into said functionally enucleated oocyte by electrofusion.

15. ~The method of any one of claims 1 to 14, wherein the method further comprises mating the non-human mammal which develops from the reconstructed embryo with a second non-human mammal to produce a transgenic offspring.

16. ~The method of any one of claims 1 to 14, wherein the transgenic non-human mammal is induced to lactate.

17. ~The method of any one of claims 1 to 14, further comprising recovering from the transgenic non-human mammal a product encoded by said recombinant nucleic acid sequence.

18. ~The method of claim 17, wherein said product is recovered from the milk, urine, hair, blood, skin, or meat of the transgenic non-human mammal.

19. ~The method of claim 18, wherein said product is a human protein.

20. ~The method of any one of claims 1 to 4, wherein said at least one recombinant nucleic acid sequence contained in said nucleus comprises a heterologous transgenic sequence under the control of a promoter.

21. ~The method of claim 20, wherein the promoter is a caprine promoter.

22. ~The method of any one of claims 1 to 21 wherein said functionally enucleated oocyte is pretreated with ethanol.

23. ~The method of any one of claims 1 to 21 wherein said functionally enucleated oocyte is activated with a calcium ionophore.

24. ~A method of producing a transgenic non-human mammal comprising:
(a) introducing a nucleus from a non-human mammalian somatic cell into a functionally enucleated oocyte, said functionally enucleated oocyte being from the same species as said somatic cell and being in the metaphase II
stage of meiotic cell division, and said nucleus from said somatic cell comprising at least one recombinant nucleic acid sequence under the control of at least one promoter sequence, to form a reconstructed embryo;
(b) transferring said reconstructed embryo to a non-human mammalian recipient when said reconstructed embryo is in the 2- to 8-cell stage of embryogenesis; and (c) allowing said reconstructed embryo to develop into a mammal, thereby providing the transgenic non-human mammal.

25. ~The method of claim 24, wherein the transferring step (b) is carried out when the reconstructed embryo is in the 2-cell stage.

26. ~The method of claim 24, wherein the transferring step (b) is carried out when the reconstructed embryo is in the 4-cell stage.

27. ~The method of claim 24, wherein the transferring step (b) is carried out when the reconstructed embryo is in the 8-cell stage.

28. ~The method of any one of claims 24 to 27 wherein said at least one recombinant nucleic acid sequence is a DNA
sequence encoding a desired gene and said at least one promoter sequence is a tissue specific promoter.

29. ~The method of claim 28, wherein said tissue-specific promoter is a promoter preferentially expressed in mammary gland epithelial cells.

30. ~The method of claim 29, wherein said promoter is selected from the group consisting of a .beta.-casein promoter, a .beta.-lactoglobin promoter, whey acid protein promoter and lactalbumin promoter.

31. ~The method of any one of claims 24 to 30, wherein said transgenic non-human mammal is a goat.

32. ~The method of any one of claims 24 to 31, wherein said at least one recombinant nucleic acid sequence encodes a polypeptide selected from the group consisting of an .alpha.-1 proteinase inhibitor, an alkaline phosphotase, an angiogenin, an extracellular superoxide dismutase, a fibrogen, a glucocerebrosidase, a glutamate decarboxylase, a human serum albumin, a myelin basic protein, a pro-insulin, a soluble CD4, a lactoferrin, a lactoglobulin, a lysozyme, a lactoalbumin, an erythropoietin, a tissue plasminogen activator, a human growth factor, an antithrombin III, an insulin, a prolactin, and an .alpha.-1-antitrypsin.

33. The method of any one of claims 24 to 32, wherein said somatic cell is selected from a group of cell types present in a non-human mammal consisting of:
a) fibroblasts b) cumulus cells c) neural cells d) mammary cells; and e) myocytes.

34. The method of claim 33, wherein the fibroblast is an embryonic fibroblast.

35. The. method of any one of claims 24 to 34, wherein the somatic cell is in G1 stage.

36. The method of any one of claims 24 to 34, wherein the somatic cell is in G0 stage.

37. The method of any one of claims 24 to 36, wherein the nucleus of said somatic cell is introduced into said functionally enucleated oocyte by electrofusion.

38. The method of any one of claims 24 to 36, wherein the method further comprises mating the non-human mammal which develops from the reconstructed embryo with a second non-human mammal to produce a transgenic offspring.