WO2008027241A2 - Non-human embryonic stem cell lines and transgenic animals derived from them - Google Patents

Abstract

Stem cells and stem cell lines, derived from animal embryos, such as from goat, are disclosed along with methods of producing such cells and cell lines. Also described are methods of using such cells, and cell lines, for the generation of transgenic animals, such as transgenic goats. Further disclosed are transgenic animals derived from such cells, and cell lines, wherein said animals comprise a heterologous transgene that expresses a selected protein, especially where said protein is produced in the milk or urine of the transgenic animal. An animal model for studying disease or for insertion of stems cells or cells derived from stem cells is also presented.

Description

NON-HUMAN EMBRYONIC STEM CELL LINES AND TRANSGENIC ANIMALS DERIVED FROM THEM

This application claims priority of U.S. Provisional Application 60/841 ,126, filed 30 August 2006, the disclosure of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of non-human mammalian embryonic stem cells and the generation of non-human transgenic mammals from stem cell lines.

BACKGROUND OF THE INVENTION

Significant genetic improvement in many domesticated animals, based on phenotypic traits, has resulted from selected breeding, including dairy cows that produce more milk and that produce milk having improved fat and protein profiles. However, many of these technologies have focused on improving genetically defined traits already extant in the animal. Conversely, use of genetic engineering techniques to produce transgenic animals takes advantage of the ability to greatly enhance traits of the subject animals or to produce animals that are sources of new traits, such as the production of valuable commercial and therapeutic products. Such animals may therefore provide a source of relatively large amounts of otherwise difficult to prepare polypeptides and proteins that are of potentially significant clinical or commercial utility. Among the techniques that have been used to create transgenic animals are pronuclear injection, commonly used for germ line insertion of genes. While this technique can be used successfully with a range of animals, but the inability to control integration of the transgene and the large number of ova which must be injected to obtain even a single transgenic offspring combine to make the technique rather inefficient for animals other than mice.

Nuclear transfer, wherein the nucleus of a donor cell is introduced into a recipient oocyte, is also useful in the generation of transgenic animals. In embryo transfer, an embryo taken from a donor animal is transferred to a recipient animal who brings it to term.

Transgenic mice have been created by genetically manipulating murine embryonic stem cells (ESC), e.g., by injecting a transgene into the ESC, and then injecting the altered embryonic stem cells into a host embryo to produce mosaics in which genetically altered cells contribute to the somatic and germ cells.

Another approach to the generation of transgenic animals is by nuclear transfer (NT), wherein the nucleus of a donor cell is introduced into a recipient oocyte, such as an enucleated oocyte. Offspring have also been reported in the bovine and sheep from cultured inner cell mass (ICM) cells and embryonic disks, respectively, using the technique of nuclear transfer.

Fetal cell populations have been used for somatic cell nuclear transfer and, while fetal cells are able to undergo between 70-80 cell divisions, most cell types undergo senescence before they can be used for nuclear transfer. For recombinant protein production, a transgenic founder animal is the most important application of somatic cell nuclear transfer. Because only primary cells can be used as nuclear donors, one challenge has been to select the transfected clones before the onset of growth arrest brought on by tissue culture-induced senescence. Other problems relate to the impact of the selection process on the ability of the recombinant cell lines to function effectively as cell donors and the possible effects of these treatments on the health of the resulting clones. In addition, one important factor making gene targeting and homologous recombination impractical, or even impossible, in large animals is the short lifespan of somatic cells. Derivation of ES cells in mice facilitated generation of many knockout mice by gene targeting as a model for several human disease. In addition, use of ES cells as donors increased the efficiency of NT production over 15-20% in mice.

Bovine animals have been cloned using embryonic cells derived from cell embryos by utilizing nuclear transfer techniques. Cloned bovine embryos were formed by nuclear transfer techniques utilizing the inner cell mass cells of a blastocyst stage embryo. Sims & First, 1993, Theriogenology 39:313 and

Keefer et al., 1994, MoI. Reprod. Dev. 38:264-268. Cloned bovine embryos have been prepared by nuclear transfer techniques that utilized PGCs isolated from fetal tissue. Delhaise et al., 1995, Reprod. Fert. Develop.

7:1217-1219; Lavoir 1994, J Reprod. Dev. 37:413-424; and PCT application

WO 95/10599 entitled "Embryonic Stem Cell-Like Cells." However, the cloned

PGC-derived bovine embryos never clearly developed past the first trimester during gestation. Similarly, embryonic stem cell-derived bovine embryos never developed past fifty-five days, presumably due to incomplete placental development. (See: Stice et al., Biol. Reprod., 54: 100-110 (1996)).

Still, there remains a great need in the art for methods and materials that increase cloning efficiency. In addition there remains a great need in the art to expand the variety of cells that can be utilized as nuclear donors, especially expanding nuclear donors to non-embryonic cells. There remains a long felt need in the art for karyotypically stable permanent cell lines that can be used for genome manipulation and production of transgenic cloned animals.

Chimeric animals in which the transgenic host cells have contributed to the tissue-type wherein the promoter of the expression construct is active (e.g., mammary gland for WAP promoter) may be used to characterize or isolate recombinant BChE and/or glucosyltransferase enzymes. More preferably, where the transgenic host cells have contibuted to the germ line, chimeras may be used in breeding schemes to generate non-chimeric offspring which are wholly transgenic.

Where transgene expression is driven by a urinary endothelium- specific promoter, urine of transgenic animals may be collected for purification and characterization of recombinant enzymes. Where transgene expression is driven by a mammary gland-specific promoter, lactation of the transgenic animals may be induced or maintained, where the resultant milk may be collected for purification and characterization of recombinant enzymes.

Embryonic stem cells have also been studied as a means of developing animal models and treatment strategies for diseases as well as to generate tissue for regenerative medical procedures and to study cell renewal and differentiation. As noted above, ES cells have been studied in mice (and even primates) but have not been conclusively established in other large mammals.

The present invention solves such problems by providing methods for generating transgenic animals, such as goats, using embryonic stem cells (ESCs), including transgenic ESCs that are capable of germ-line transmission of the transgene. The goat is believed to represent a good animal model for ES studies because its embryogenesis is well understood and permits isolation of ES cells and the following of functional endpoints not yet available for large primates. Thus, there is as yet an unmet need for the development of a non-rodent model for germ cell differentiation of pluripotent cells that allows for definite molecular studies and differentiation endpoints, such as the formation of neural cells and other specific differentiated cells, especially those intimately involved in the disease process. Thus, in accordance with the present invention there are developed methods for producing goat ES cells and cell lines that show pluripotency and that can be maintained in an undifferentiated state for over 100 passages. Also in accordance with the present invention, such cells are pluripotent both in vitro and in vivo. BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated embryonic stem cell derived from non-human mammals, for example, a goat, preferably having the capacity for germ-line transmission. In one embodiment thereof, such isolated embryonic stem cell is derived from the inner cell mass of a goat. In another embodiment, said embryonic stem cell is derived from the embryonic disc or epiblast layers of a goat. The present invention includes embodiments wherein the goat is a dwarf goat or wherein the goat is a

Saanen and Saanen-Cross goat.

The present invention also provides an isolated embryonic stem cell, preferably a goat embryonic stem cell (gESC) that is positive for markers characteristic of mammalian stem cells, including but not limited to alkaline phosphatase, Oct-4, SSEA-1 and SSEA-4. In one embodiment, said gESC exhibits all 4 of the latter markers.

In another aspect, the embryonic stem cell, preferably a gESC, of the present invention is a transfected (i.e., transfected with a transgene or heterologous DNA encoding a protein) embryonic stem cell. In one embodiment, such recombinant embryonic stem cell comprises one or more transgenes. Specific non-limiting examples of such embryonic stem cells include those wherein the transgene comprises a DNA sequence or construct encoding a selected polypeptide or protein, preferably an enzyme, such as butyrylcholiπesterase (BChE), said DNA sequence being in operable linkage with a selected promoter. In one embodiment, said polypeptide is a fusion polypeptide, preferably a polypeptide fused to the amino acid sequence for human serum albumin (HSA).

In non-limiting embodiments of such constructs, the promoter is a tissue-specific promoter, such as a mammary gland-specific promoter (for example, a WAP (whey acidic protein) or a casein promoter) or a urinary endothelium-specific promoter (for example, a uroplakiπ promoter or a uromodulin promoter). The promoter portion of such genetic construct may also be a ubiquitous promoter, such as the cytomegalovirus (CMV) promoter.

In another aspect of the present invention, the polypeptide or protein encoded by said genetic construct or nucleotide sequence is a protein or polypeptide of choice. In one such embodiment, said protein is butyrylcholinesterase (BChE), such as human butyrylcholinesterase (hBChE), preferably a recombinant hBChE).

In a further aspect, the present invention provides a method for producing a goat, comprising:

(a) introducing a nucleus from a goat embryonic stem cell (gESC) into an enucleated goat oocyte to form a zygote, (b) transplanting said zygote into a pseudopregnant goat, and

(c) allowing the resulting embryo to develop to term.

In one embodiment of the foregoing, said gESC is a transfected gESC. In another embodiment, said nucleus of step (a) is part of a gESC. Such nucleus may thus be transferred by nuclear transfer procedures or by fusion of an oocyte, such as an enucleated oocyte, with a gESC of the invention.

In a further aspect, the present invention provides a method for producing a transgenic non-human mammal, for example, a goat, whose somatic and germ cells contain a transgene, comprising:

(a) introducing a non-human mammalian embryonic stem cell, such as a goat embryonic stem cell, for example, a dwarf or other type of goat as described herein, into an enucleated oocyte (to form a zygote, i.e., a fertilized oocyte, or embryo) of a non-human mammal, wherein said embryonic stem cell contains a transgene comprising a DNA sequence or genetic construct encoding a polypeptide or protein and is in operable linkage with a suitable promoter, (b) transplanting said zygote or embryo into a pseudopregnant non- human mammal, such as a pseudopregnant goat, including a dwarf goat,

(c) allowing the resulting embryo to develop to term, and

(d) identifying at least one non-human mammal, for example, a goat, such as a dwarf goat, wherein, in separate preferred embodiments the expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue or urinary tissue of said non-human mammal, for example, a goat, including a dwarf goat.

In one such embodiment, said stem cell is introduced by fusion of the stem cell with said oocyte.

In another aspect, the present invention provides a method for producing a transgenic non-human mammal, for example, a goat, such as a dwarf goat, whose somatic and germ cells contain a transgene, comprising:

(a) introducing a nucleus or nuclear material from a non-human mammalian embryonic stem cell, such as a goat embryonic stem cell, for example, a dwarf goat, into a mammalian cell, such as an enucleated oocyte (to form a zygote or re-constructed embryo) of the same or different species of non-human mammal, wherein said embryonic stem cell nucleus contains a transgene comprising a DNA sequence or genetic construct encoding a polypeptide or protein of choice, especially a heterologous polypeptide or protein,- that is in operable linkage with a suitable promoter,

(b) transplanting said zygote or embryo into a pseudopregnant non- human mammal, such as a pseudopregnant goat, including a dwarf goat,

(c) allowing the resulting embryo to develop to term, and

(d) identifying at least one non-human mammal, for example, a goat, especially a dwarf goat, wherein in separate preferred embodiments the expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue or urinary tissue of said non-human mammal, for example, a goat, including a dwarf goat.

In one such embodiment, said nucleus is introduced by fusion of the stem cell with said oocyte. The present invention also relates to such methods that further comprise breeding a non-human mammal, such as a goat, for example, a dwarf goat, to produce a transgenic non-human mammal, such as a transgenic goat, wherein expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue of said dwarf goat.

Ih non-limiting embodiments of the methods of the invention, the non- human mammal is preferably a goat.

In another aspect, the present invention provides transgenic animals, preferably transgenic goats, produced by any of the methods of the invention.

The present invention also provides for the production of animal models for the study of disease, such as diseases of the nervous system, for example, diseases of the central nervous system. In one non-limiting embodiment, there is provided a transgenic goat whose nervous system produces a transgenic mutated protein, which protein causes aberrant functioning of neurons or other cells of the nervous system, for example, where the protein is some type of receptor. Such animal model is thus available for study of the effects of such proteins as well as for the testing of potential agents for treating such diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows goat in vivo derived blastocysts cultured in vitro for several' days. Here, gES cells were derived from goat in wVo-derived blastocysts. A: in wVo-derived blastocysts after 2 days in culture. B: Floating blastocyst after 5 days in culture. The prominent ICM disc or epiblast, a pluripotent derivative of the inner cell mass, is surrounded by trophectodermal cells. C: ICM/epiblast cells isolated from blastocyst after 5 days in culture. D: gES cell colony on feeder cells 7 days after seeding. Bar = 100 μm.

Figures 2A - 2F show goat ES cells differentiated to form ectodermal cells in vitro. Fig. 2A shows a gES cell colony at passage >35 without feeder cells, showing the beginning of formation of an embryoid body. Fig. 2B shows differentiated gES cells exhibiting epithelial cell-like morphology. Fig. 2C shows embryoid bodies formed from gES cells cultured in suspension. Fig. 2D shows formation of neurosphere-like structures from EBs (embryoid bodies). Fig. 2E. shows a differentiating gES colony forming nestin. Fig. 2F shows a cell with a neuron-like morphology generated from gES cell differentiation following 5 to 8 days in culture. Scale bars represent 50 μm in Fig. 2B and

100 μm in the others.

Figure 3 shows that gES cells as early as passage 6 expressed markers for non-differentiated pluripotent cells. Fig. 3A shows expression of alkaline phosphatase (ALP) goat ES-like cells, Fig. 3B shows goat ES cells stained strongly positive for Oct-4 (Oct-4 Promoter (i.e., Octamer-4) is a transcription factor of the POU family and is critically involved with self- renewal of undifferentiated embryonic ES cells; it is frequently used as a marker for undifferentiated ES cells (mouse ES cells were used as a positive control and also stained for Oct-4)); Fig. 3C shows ES cells stained positive for SSEA-4; and Fig. 3D shows ES cells stained positive for SSEA-1.

Figure 4 shows goat ES cells transfected with the CEeGFP plasmid.

Figs. 4A and 4B show transfected gES cells after trypsinization at passage 79 and Fig. 4C shows transfected gES cells (eGFP-gES cells) growing on a goat fetal fibroblast feeder layer.

Figures 5A and 5B show gES cells transfected with GFP aggregating on feeder cells. ^" Figure 6A shows results of a PCR analysis of gES cells for the presence of the monomeric human butyrylcholinesterase (hBChE) transgene (Nex91-B-casein primers). Lane 1 is a 100 bp ladder, lanes 2-9 show 8 gES clones, lanes 10 and 11 show gESC 15AA passage 77 and 82, respectively, (non-transfected) cell, lane 12 is BChE plasmid DNA alone, lane 13 is DNA from a BChE transgenic goat, containing dimeric BChE, and lane 14 is a water blank. Fig. 6B shows results of a PCR analysis of gES cells for the insulator surrounding the transgene (top band) and a region of endogenous β- casein (bottom band). Here, Lane 1 is a 100 bp ladder, lanes 2-3 are blanks, lanes 4-11 show 8 gES clones transfected with the hBChE plasmid, lanes 12 and 13- show respectively a gES 15AA passage 77 (non-transfected) and passage 82 (non-transfected) cell, lane 14 is BChE plasmid DNA alone, lane 15 is a blank lane and lane 16 is DNA from a BChE transgenic goat.

DEFINITIONS

As used herein, the following terms have the indicated meaning unless expressly stated otherwise.

"Embryonic stem (ES) cells" refers to cells derived from totipotent cells of the early mammalian embryo and such ES cells are capable of unlimited and undifferentiated proliferation in vitro. Some cells require various factors to be kept in culture media in an undifferentiated state, such as the cytokine, Leukemia Inhibitory Factor (LIF). Thus, undifferentiated mouse ES cells begin to differentiate into cells of all 3 germ layers when LIF is removed from the medium. Other ES cells, like human cells, cannot be maintained using LIF but often need feeder cell layers for self-renewal.

The term "inner cell mass" as used herein refers to the cells that gives rise to the embryo proper. The cells that line the outside of a blastocyst are referred to as the trophoblast of the embryo. The methods for isolating inner cell mass cells from an embryo are well known to a person of ordinary skill in the art. See, Sims and First, 1993, Theriogenology 39:313; and Keefer et al., 1994, MoI. Reprod. Dev. 38:264-268, hereby incorporated by reference herein in their entireties, including all figures, tables, and drawings. The term "pre- blastocyst" is well known in the art and is referred to previously.

The term "gamete" as used herein refers to any cell participating, directly or indirectly, to the reproductive system of an animal. Examples of gametes are spermatocytes, spermatogonia, oocytes, and oogonia. Gametes can be present in fluids, tissues, and organs collected from animals (e.g., sperm is present in semen). For example, methods of collecting semen for the purposes of artificial insemination are well known to a person of ordinary skill in the art. See, e.g., Physiology of Reproduction and Artificial Insemination of Cattle (2nd edition), Salisbury et al., copyright 1961 , 1978, WH Freeman & Co., San Francisco. However, the invention relates to the collection of any type of gamete from an animal.

The term "butyrylcholinesterase enzyme" or "BChE enzyme" means a polypeptide capable of hydrolizing acetylcholine and butyrylcholine, and whose catalytic activity is inhibited by the chemical inhibitor iso-OMPA. Preferred BChE enzymes to be produced by the present invention are mammalian BChE enzymes. Preferred mammalian BChE enzymes include human BChE enzymes. The term "BChE enzyme" also encompasses pharmaceutically acceptable salts of such a polypeptide.

The term "recombinant butyrylcholinesterase" or "recombinant BChE" means a BChE enzyme produced by a transiently transfected, stably transfected, or transgenic host cell or animal as directed by one of the expression constructs of the invention. The term "recombinant BChE" also encompasses pharmaceutically acceptable salts of such a polypeptide.

The term "genetically-engineered DNA sequence" means a DNA sequence wherein the component sequence elements of the DNA sequence are organized within the DNA sequence in a manner not found in nature. Such a genetically-engineered DNA sequence may be found, for example, ex vivo as isolated DNA₁ in vivo as extra-chromosomal DNA, or in vivo as part of the genomic DNA.

The term "expression construct" or "construct" or "genetic construct" means a nucleic acid sequence comprising a target nucleic acid sequence or sequences whose expression is desired, operably linked to sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) within the chosen host cells. Such sequence elements may include a promoter, a signal sequence for secretion, a polyadenylation signal, intronic sequences, insulator sequences, and other elements described in the invention. The "expression construct" or "construct" may further comprise "vector sequences". The term "vector sequences" means any of several nucleic acid sequences established in the art which have utility in the recombinant DNA technologies of the invention to facilitate the cloning and propagation of the expression constructs including (but not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.

The term "bi-cistronic construct" means any construct that provides for the expression of two independent translated products. These two products may translated from a single mRNA encoded by the bi-cistronic construct or from two independent mRNAs where each of the mRNAs is encoded within the same bi-cistronic construct. The term "poly-cistronic construct" means any construct that provides for the expression of more than two independent translated products.

The term "operably linked" means that a target nucleic acid sequence and one or more regulatory sequences (e.g., promoters) are physically linked so as to permit expression of the polypeptide encoded by the target nucleic acid sequence within a host cell.

The term "signal sequence" means a nucleic acid sequence which, when incorporated into a nucleic acid sequence encoding a polypeptide, directs secretion of the translated polypeptide (e.g., a BChE enzyme and/or a glycosyltransferase) from cells which express said polypeptide. The signal sequence is preferably located at the 5' end of the nucleic acid sequence encoding the polypetide, such that the polypeptide sequence encoded by the signal sequence is located at the N-terminus of the translated polypeptide. The term "signal peptide" means the peptide sequence resulting from translation of a signal sequence.

The term "mammary gland-specific promoter" means a promoter that drives expression of a polypedtide encoded by a nucleic acid sequence to which the promoter is operably linked, where said expression occurs primarily in the in the mammary cells of the mammal, wherefrom the expressed polypeptide may be secreted into the milk. Preferred mammary gland-specific promoters include the β-casein promoter and the whey acidic protein (WAP) promoter

The term "urinary endothelium-specific promoter" means a promoter that drives expression of a polypedtide encoded by a nucleic acid sequence to which the promoter is operably linked, where said expression occurs primarily in the endothelial cells of the kidney, ureter, bladder, and/or urethra, wherefrom the expressed polypeptide may be secreted into the urine. The term "urothelium" or "urothelial cells" refers to the endothelial cells forming the epithelial lining of the ureter, bladder, and urethra.

The term "host cell" means a cell which has been transfected with one or more expression constructs of the invention. Such host cells include mammalian embryonic stem cells in in vitro culture and cells found in vivo in an animal.

The term "transfection" means the process of introducing one or more of the expression constructs of the invention into a host cell by any of the methods well established in the art, including (but not limited to) microinjection, electroporation, liposome-mediated transfection, calcium phosphate-mediated transfection, or virus-mediated transfection. A host cell into which an expression construct of the invention has been introduced by transfection is "traπsfected". The term "transiently transfected cell" means a host cell wherein the introduced expression construct is not permanently integrated into the genome of the host cell or its progeny, and therefore may be eliminated from the host cell or its progeny over time. The term "stably transfected cell" means a host cell wherein the introduced expression construct has integrated into the genome of the host cell and its progeny.

The term "transgene" means any segment of an expression construct of the invention which has become integrated into the genome of a transfected host cell. Host cells' containing such transgenes are "transgenic". Animals composed partially or entirely of such transgenic host cells are "transgenic animals". Preferably, the transgenic animals are transgenic mammals other than human (e.g., rodents or ruminants). Animals composed partially, but not entirely, of such transgenic host cells are "chimeras" or "chimeric animals". Chimeric animals originate from two different zygotes whereas partially transgenic animals are called "mosaics."

By "signal peptide," is meant a polypeptide which facilitates secretion of the protein to which it is linked. The signal peptide can be naturally occurring in the heterologous protein (e.g., signal peptides of naturally secreted proteins such as human placental β-galactosidase, β-galactosidase of Aspergillus niger, and hGH). Alternatively, the genetic construct can be engineered so that a signal peptide is bonded to the heterologous protein.

By "heterologous" protein, gene product, nucleic acid, or sequence, is meant a protein, gene product, nucleic acid, or sequence which is introduced into a ruminant mammary epithelial cell. For example, a human or bacterial protein or gene product is heterologous to a ruminant mammary epithelial cell.

In addition, a protein or gene product that is not naturally present in a mammary epithelial cell of a ruminant, but which is naturally present in another cell of the ruminant (e.g., insulinlike growth factor, insulin, or follicle stimulating hormone), is heterologous to the mammary epithelial cell. Also included, is a protein or gene product that is naturally present in a ruminant mammary epithelial cell, but which is expressed from a genetic construct which is not naturally present in the mammary epithelial cell (e.g., milk proteins such as β-casein and lactoferrin expressed from a plasmid). Expression of such a protein or gene product from a genetic construct can elevate the level of the protein or gene product in the mammary epithelial cell and/or milk. The transgenes used in the invention encode such heterologous proteins.

"Surface Stage embryonic antigen" (SSEA-1 and SSEA-4) refers to a glycolipid carbohydrate epitope present on undifferentiated ES cells. It is a cell-surface marker for ES cells that can be used to characterize pluripotent stem cells. SSEA-1 is a fucosylated derivative of type 2 polylactosamine that appears during late cleavage stages of, for example, mouse embryos and is strongly expressed by undifferentiated, murine ES cells. When they differentiate, murine ES cells lose SSEA-1 expression and may be accompanied, in some instances, by the appearance of other markers like SSEA-3 and SSEA-4. In contrast, human embryonic stem cells typically express SSEA-3 and SSEA-4 but not SSEA-1 , while the differentiation of such cells is characterized by down regulation of SSEA-3 and SSEA-4 and an up regulation of SSEA-1.

"Octamer-4 (Oct-4)" refers to a transcription factor of the POU family that is useful in characterizing the undifferentiated state of ES cells, which often exhibit a high level of expression of this factor. This protein is critically involved with self-renewal of undifferentiated embryonic stem cells. This relationship between Oct-4 and pluripotency makes it a frequest marker for pluripotent stem cells. Undifferentiated human and murine pluripotent Embryonic Stem (ES) also express Oct-4, as well as murine Embryonic Germ (EG) cells. Following stem cell differentiation, the level of Oct-4 expression decreases.

By "dwarf goat" is meant a Nigerian Dwarf goat or a Pygmy goat or any other goat of small size comparable to that of a Nigerian Dwarf goat or a

Pygmy goat. Suitable goat breeds preferably weigh approximately 80 lbs or less at maturity and weigh 2.0 kg, more preferably 1.7, or 1.5 kg at birth. Suitable breeds are of a fetal size and neonatal size which permit a non-dwarf goat (i.e., a standard goat) to which the dwarf goat embryo or zygote are transferred to bear 3 or 4, more preferably 5 or 6, dwarf goats in a single pregnancy. Achondroplastic dwarf goats are suitable for use in the method of the invention as are dwarf goats whose small stature is due to some other cause.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides established goat embryonic stem cell- like lines derived from in vivo and in vitro produced blastocyst-stage embryos. Also provided are Goat Embryonic Stem Cells (GEC) under conditions that allow the cells, or cells derived from them, to be utilized in nuclear transfer technology or other techniques for increasing the efficiency of producing transgenic animals. The disclosed methods herein have generated a goat ES cell capable of undergoing an unlimited number of symmetrical divisions without differentiating (long-term self-renewal).

The lack of ES cells presents a considerable barrier to gene targeting in livestock. One of the significant challenges to be overcome for efficient somatic cell transfection is the limited lifespan of primary cells in culture. It has been estimated that a total of about 45 population doublings are required to generate targeted cells from fetal tissue using electroporation for drug selection and high throughput screening. However, this is within the lifespan of goat fibroblast cells. In accordance with the present invention, goat ESCs have been generated that have undergone as many as 340 cell divisions. The methods of the invention have also successfully transfected these cells to show that gESCs (goat embryonic stem cells) can survive transfection treatment and are available for use in transgenic production. In general, data suggest that the transfection and selection treatment do not harm the ability of the cell line to serve as nuclear transfer donors. In accordance with the present invention, gESCs were transfected with two constructs comprising green fluorescent protein (GFP) and human butyrylcholinesterase (hBCHE) using mammary gland expression vectors. Polymerase chain reaction (PCR) was used to confirm the presence and integrity of the hBCHE transgene (Figure ^' 6). A goat ESC line transfected with GFP was verified using Fluorescence microscopy (Figures 4 and 5).

The goal of the invention is to produce a transgenic non-human mammal, especially a transgenic goat, preferably a transgenic dwarf goat, and thus provides non-limiting methods that include (a) introducing a transgene into an embryo of a dwarf goat, (b) transplanting the embryo into a pseudopregnant non-dwarf goat, and (c) allowing the embryo to develop to term. The transgene is introduced into the embryo by introducing a nucleus of a stem cell, preferably a fetal stem cell, from said goat into an enucleated oocyte or fusing such stem cell with said oocyte or introducing the stem cell itself into the oocyte and then transplanting the resulting zygote or embryo into the pseudopregnant animal.

Preferred embodiments include subsequent breeding of the offspring to produce, for example, a transgenic dwarf goat. In other preferred embodiments the introducing of the transgene into the embryo is by introducing an embryonic stem cell containing the transgene into the embryo.

In specific embodiments using dwarf goats, at least four zygotes can be transplanted into the pseudopregnant dwarf goat.

Because several dwarf goat embryos can be implanted in a single pseudo-pregnant standard goat, the methods of the invention allow one to decrease the number of recipient animals required for the production of transgenic goats. Transfer of multiple dwarf embryos to standard goats as recipients results in an increase (e.g., 2- to 4-fold) in the number of offspring per recipient, compared to the implantation of standard goat embryos into standard goat recipients. This represents a significant increase in production efficiency and a significant decrease in cost of recipient animals compared to other systems, such as the transfer of standard goat embryos to standard goat recipients. Moreover, dwarf goats have characteristics, including lack of seasonality, early onset of sexual maturity, and small fetal and neonatal size, and good milk yield, which are highly desirable in transgenic animals used for the production of pharmaceuticals or nutriceuticals in milk. Lack of seasonality and early onset of sexual maturity decrease the generation interval as compared to other dairy ruminants. Using the method of the invention, a heterologous gene product can be expressed in the milk of a transgenic animal within a time-frame which is the shortest of any dairy ruminant (e.g., a year before that of transgenic dairy cattle). Such methods are further described in U.S. Patent No. 5,907,080.

In spite of the tremendous improvement of production of large animal transgenics using somatic cell nuclear transfer (SCNT), such a technique is still an inefficient procedure. Many observed problems are believed to be caused by inadequate genomic reprogramming of the donor cells (somatic cells), as reported in mouse embryos reconstructed by SCNT using ES cells with 10-20 fold higher efficiency. Using goat ES cells in SCNT achieves an improved method and shortens the time, increases the efficiency and reduces the cost of generating the first-generation (founder) transgenic animals, a critical early step in the transgenic production process.

Furthermore, the availability of ES like cells allows targeting of genes of interest under the influence of endogenous promoters and provides for the functionality of the transgene linked to an available promoter. In accordance with the foregoing, GES-like cells from in vivo derived goat embryos were generated for the first time by the methods herein. The ES marker staining pattern is similar to ES cells derived form bovine and mouse embryos, although SSEA-4 was positive in goat ES but negative in mouse and variable in bovine. The differentiation of the derived GES resembling various somatic cell types (neuronal, epithelial etc.) is indicative of the pluripotency of the derived GES cells. These are characterized by their capacity for self-renewal and their capability to differentiate into a broad spectrum of cell types and such goat ES cells are differentiated to oocyte like structures. In addition, ES cell culture on gelatinizes dish are differentiating to neuron and astoryctes cells after passage 33. In accordance with the foregoing, pluripotent embryonic stem cells (ESC) derived, for example, from the inner cell mass (ICM) of mammalian blastocysts provide an unlimited number numbers of cells that can be used in gene targeting and are of great value to agriculture and medicine. Prior to the present disclose, embryonic stem cells with the capacity for germ line transmission have been verifiably produced only in mice and human despite many efforts to derive ESCs from other mammalian species.

Methods for the derivation, propagation and differentiation of ESCs from domestic animals have not previously been fully established. Thus, it is one object of the present invention to provide methods for the generation and characterization of embryonic stem cells, for example, goat embryonic stem cells (GESC) derived from embryos, such as in vivo produced embryos, for example, those in the blastocyst stage.

The present invention provides not only for embryonic stem cells but also provides methods for the production of transgenic non-human mammals, such as goats, including dwarf goats. Such methods of the invention serve to increase the efficiency and reduce the cost of generating founders in large animals, which is a critical early step in the transgenic production process. In addition, the embryonic stem cells provided by the present invention allow targeting of genes of interest under the influence of selected endogenous promoters of choice.

In one aspect, the present invention provides an isolated embryonic stem cell derived from mammals, for example, a goat, and having the capacity for germ-line transmission. In one embodiment thereof, such isolated embryonic stem cell is derived from the inner cell mass of a goat. In another embodiment, said embryonic stem cell is derived from the embryonic disc of a goat. The present invention includes embodiments wherein the goat is a dwarf goat.

The isolated embryonic stem cell provided herein is commonly positive for markers characteristic of mammalian stem cells, including alkaline phosphatase, Oct-4, SSEA-1 and SSEA-4 (see Figure 3).

In another aspect, the embryonic stem cell of the present invention is a recombinant embryonic stem cell. In one embodiment, such recombinant embryonic stem cell comprises one or more transgenes. Specific non-limiting examples of such embryonic stem cells include those wherein the transgene comprises a DNA sequence or construct encoding a selected polypeptide or protein, said DNA sequence being in operable linkage with a selected promoter.

By introducing a transgene into the embryonic stem cell of the invention, such transgene can be transmitted through the transgenic animal by germ-line transmission to produce additional offspring exhibiting production of a heterologous protein encoded by said transgene. The transgene introduced into the stem cell is represented by a genetic or expression construct as defined herein.

In one embodiment the genetic construct (such as a plasmid) also includes a transcription termination region, which might be a polyadenylation signal or termination regions known to affect mRNA stability (for example, those derived from the bovine growth hormone gene, globin genes, the SV40 early region or milk protein genes).

Optionally, the genetic construct also includes an intron region that can increase the level of expression of the transgene. Generally, the intron should be placed between the transcription initiation site and the translational start codon, or 3' of the translational stop codon, or within the coding, or exon, region of the transgene. A useful intron region will also commonly contain a 5¹ splice site (i.e., a donor site), a 3' splice site (i.e., an acceptor site), and at least 100 nucleotides between the two sites. In one embodiment the intron is selected from those naturally found in genes of goats (for example, genes that express caseins or other milk proteins).

The genetic constructs useful in the invention are prepared using conventional techniques for DNA isolation. Such DNA is commonly free of endotoxins. Any of the approved methods for purifying DNA for use in humans (e.g., a Qiagen DNA extraction kit and endotoxin elimination kit) may be employed without limiting the present invention. Such constructs can be further characterized by DNA sequencing and creation of restriction maps to yield information regarding the orientation and arrangement of the gene encoding the selected heterologous protein relative to the other components of the vector.

Choice of a heterologus protein for production in a transgenic ESC or animal will not limit the invention. Thus, any protein conveniently produced in a dwarf goat is suitable for use in the methods disclosed herein. In most cases, the protein of choice will be of research or commercial, including therapeutic, value. These include BChE₁ htPA, hGH, and IL-6. Also useful are proteins that increase the nutritional value of animal products, such as milk (e.g., β-casein and lactoferrin). Many genes encoding these and other useful proteins have been identified and cloned, allowing them to be readily subcloned for use in the production of transgenic dwarf goats.

Many proteins of value in food science and technology can be produced transgenically using the methods of the invention and the animals produced by such methods. These include those possessing enzymatic activity directed toward a component of milk; which can be used to alter the lipid, protein, or carbohydrate content of the milk. For example, β- galactosidase can be made by transgenic animals of the invention to yield milk having a reduced lactose level. Genes encoding β-galactosidase can be derived from any of a number of organisms, including Aspergillus niger, (Kumar et al., 1992, Bio/technology 10:82); Homo sapiens (Oshima et al., 1988, Biochem. Biophys. Res. Comm. 157:238); Kluyveromyces lactis (U.S. Pat. No. 5,047,340; Sreekrishna and Dickson, 1985, Proc. Natl. Acad Sci. 82:7909; and Poch et al., 1992, Gene 118:55); Lactobacillus bulgaricus (Schmidt et al., 1989, J. Bacteriology 171 :625).

Other proteins that can be produced is desirable quantities by the transgenic animals produced according to the invention include tissue plasminogen activator (e.g., human tissue plasminogen activator (htPA)), cytokines (e.g., an interleukin such as IL-6 or IL-2), aspartic proteases (e.g., aspartic proteases from Rhizomucor nichei or Rhizomucor puscillus), lysozyme, stearyl-CoA desaturases, lipases (e.g., a bile-activated lipase or carboxy ester lipase), galactosyltransferases, one of the blood clotting factors and hormones (including factor I, II, III, IV, V, VII, VIII, IX, X, Xl, Xl, or XIII)₁ growth factor (e.g., human growth hormone (hGH), epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor, nerve growth factor, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, or erythropoietin), oncoproteins (e.g., ras, fos, jun, myc, kit, or myb), a tumor suppressor protein (e.g., p53), a milk protein (e.g., β-casein or lactoferrin), peptide hormones (e.g., insulin or follicle-stimulating hormone) and receptors (e.g., an insulin receptor), translation factors (e.g., eukaryotic initiation factor 4E), transcription factors (e.g., mammary gland specific factor), protein C, al- antitrypsin, urokinase plasminogen activator, human serum albumin, cystic fibrosis transmembrane conductance regulator, gamma-interferon, human CD4, or erythropoietin. In one embodiment, the protein expressed by the transgene is the human form of the protein. .

Depending on the uses to which the transgenic protein, or heterologous protein, is to be put, the gene encoding this protein can be mutated. Such mutations may also affect the ability to generate the protein.

For example, mutations in the 5'- or 3'-untranslated regions of the gene can improve expression of the gene encoding the heterologous protein. Other useful mutations or deletions are those which increase secretion of the protein from the cell. Sequences encoding endoplasmic reticulum retention signals or other sorting inhibitory signals can be deleted from the genetic construct or mutated to be non-functional. In addition, truncated versions of naturally- occurring proteins can be used in the invention if it possesses a useful biological activity.

Each transgenic protein produced according to the invention should be bonded to a signal peptide if the protein is to be secreted (for example, from the mammary epithelial cell). The signal peptide can be a naturally-occurring component of the heterologous protein (e.g., the signal peptide of human placental β-galactosidase). Where the heterologous protein is not naturally a secreted protein, if secretion is desired, the genetic construct should be assembled such that a signal peptide is bonded to the heterologous protein so that the signal peptide directs secretion of the protein from the cell. Useful signal peptides can be derived from genes such as casein genes, or the gene for human alkaline phosphatase.

In non-limiting embodiments of such constructs, the promoter is a tissue-specific promoter, such as a mammary gland-specific promoter (for example, a WAP (whey acidic protein) or a casein promoter) or a urinary endothelium-specific promoter (for example, a uroplakin promoter or a uromodulin promoter).

Useful promoters for the expression of transgenes in the mammary tissue include promoters which naturally drive the expression of mammary- specific. For example, the αS1 -casein promoters, αS2-casein promoters, β- casein promoters, κ-casein promoters, β-lactoglobulin promoters, whey acidic protein promoters, and α-lactalbumin promoters can be used. If desired, the promoter can be operably linked to one or more enhancer elements such that the enhancer elemeήt(s) increases transcription of the gene encoding the heterologous gene product. For specific expression in the mammary tissue of transgenic animals, the promoter sequences may be derived from a mammalian mammary- specific gene. Examples of suitable mammary-specific promoters include: the whey acidic protein (WAP) promoter [U.S. Pat. Nos. 5,831 ,141 and 6,268,545, Andres, et al. Proc Natl Acad Sci USA (1987) 84(5):1299-1303], α~S1-casein [U.S. Pat. Nos. .5,750,172 and 6,013,857, PCT publication Nos. WO91/08216 and WO93/25567], αS2-casein, β-casein [U.S. Pat. No. 5,304,489; Lee, et al. Nucleic Acids Res. (1988) 16:1027-1041], κ-casein [Baranyi, et al. Gene (1996) 174(1):27-34; Gutierrez, et al. Transgenic Research (1996) 5(4):271- 279], β-lactoglobin [McClenaghan, et al. Biochem J (1995) 310(Pt2):637-641], and α-lactalbumin [Vilotte, et al. Eur. J. Biochem. (1989) 186: 43-48; PCT publication No. WO88/01648].

For specific expression in the urinary endothelium of transgenic animals, the promoter sequences may be derived from a mammalian urinary endothelium-specific gene. Examples of suitable urinary endothelium-specific promoters include the uroplakin Il promoter [Kerr, et al. Nature Biotechnology . (1998) 16(1 ):75-79], and the uromodulin promoter [Zbikowska, et al. Biochem J (2002) 365(Pt1):7-1 1; Zbikowska, et al. Transgenic Res 2002 11(4):425- 435].

The promoter portion of such genetic construct may also be a ubiquitous promoter, such as the cytomegalovirus (CMV) promoter.

Promoter sequences for ubiquitous expression may include synthetic and natural viral sequences [e.g., human cytomegalovirus immediate early promoter (CMV); simian virus 40 early promoter (SV40); Rous sarcoma virus (RSV); or adenovirus major late promoter] which confer a strong level of transcription of the nucleic acid molecule to which they are operably linked. The promoter can also be modified by the deletion and/or addition of sequences, such as enhancers (e.g., a CMV, SV40, or RSV enhancer), or tandem repeats of such sequences. The addition of strong enhancer elements may increase transcription by 10-100 fold. In another aspect of the present invention, the polypeptide or protein encoded by said genetic construct or nucleotide sequence is a protein or polypeptide of choice. In one such embodiment, said protein is butyrylcholinesterase (BChE), such as human butyrylcholinesterase (hBChE).

In a further aspect, the present invention provides a method for producing a goat, comprising:

(a) introducing a nucleus from a goat embryonic stem cell (gESC) into an enucleated goat oocyte to form a zygote,

(b) transplanting said zygote into a pseudopregnant goat, and

(c) allowing the resulting embryo to develop to term.

In one embodiment of the foregoing, said gESC is a transfected gESC. In another embodiment, said nucleus of step (a) is part of a gESC. Such nucleus may thus be transferred by nuclear transfer procedures or by fusion of an oocyte, such as an enucleated oocyte, with a gESC of the invention.

In one aspect, the present invention provides a method for producing a transgenic non-human mammal, for example, a goat, such as a dwarf goat, whose somatic and germ cells contain a transgene, comprising:

(a) introducing a nucleus or nuclear material from a non-human mammalian embryonic stem cell, such as a goat embryonic stem cell, for example, a dwarf goat, into a mammalian cell, such as an enucleated oocyte (to form a zygote, which is a fertilized oocyte) of the same or different species of non-human mammal, wherein said embryonic stem cell nucleus contains a transgene comprising a DNA sequence or genetic construct encoding a polypeptide or protein of choice, especially a heterologous polypeptide or protein, that is in operable linkage with a suitable promoter, (b) transplanting said zygote or fertilized oocyte into a pseudopregnant non-human mammal, such as a pseudopregnant goat, including a dwarf goat, (c) allowing the resulting embryo to develop to term, and (d) identifying at least one non-human mammal, for example, a goat, especially a dwarf goat, wherein , in separate preferred embodiments the expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue or urinary tissue of said non-human mammal, for example, a goat, including a dwarf goat.

In a further aspect, the present invention provides a method for producing a transgenic non-human mammal, for example, a goat, such as a dwarf goat whose somatic and germ cells contain a transgene, comprising: (a) introducing a non-human mammalian embryonic stem cell, such as a goat embryonic stem cell, for example, a dwarf goat, into an enucleated oocyte or ovum of a non-human mammal, wherein said embryonic stem cell contains a transgene comprising a DNA sequence or genetic construct encoding a polypeptide or protein and is in operable linkage with a suitable promoter,

(b) transplanting said zygote or fertilized ovum into a pseudopregnant non-human mammal, such as a pseudopregnant goat, including a dwarf goat,

(c) allowing the resulting embryo or implanted zygote to develop to term, and (d) identifying at least one non-human mammal, for example, a goat, such as a dwarf goat, wherein , in separate preferred embodiments the expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue or urinary tissue of said non-human mammal, for example, a goat, including a dwarf goat.

In one embodiment of such method, the embryonic stem cell is the embryonic stem cell of the invention. For purposes of the invention, the term "embryo" may include a zygote.

The present invention also relates to a transgenic dwarf goat produced by the above method. The transgenic dwarf goats produced by the method of the invention can be used to produce useful human therapeutic proteins (e.g., human growth hormone) and veterinary therapeutic proteins (e.g., IL-6) in the milk of the dwarf goats. Production of the heterologous protein in a mammal facilitates post-translational modification of the protein and obviates expensive cell culture media used in in vitro methods of protein production. The invention also offers the advantage that the heterologous protein can be produced in large quantities. Transgenic goats can also be used to alter the characteristics of milk.

Transgenic goats can be used for many of the same purposes for which other transgenic animals have been used. The following references describe a variety of uses for transgenic animals: Sarvetnick et al. (PCT Application No. PCT/US94/04708); Bjursell et al. (PCT Application No. PCT/SE93/00515); Lonberg (PCT Application No. PCT/US94/04580); and Abraham et al. (PCT Applicaton No. PCT/GB94/00569).

In one embodiment of this, the embryonic stem cell is the embryonic stem cell of the invention.

In non-limiting embodiments of the present invention, the pseudopregnant goat is a non-dwarf goat.

The present invention also relates to such methods that comprise breeding a non-human mammal, such as a dwarf goat, to produce a transgenic non-human mammal, such as a transgenic dwarf goat, wherein expression of said transgene results in the production and secretion of a protein encoded by said DNA in the mammary tissue of said dwarf goat.

For example, expression of an appropriate transgene can cause alterations in the protein, lipid, or carbohydrate content of the milk. Useful milk products, such as those having a reduced lactose content, can readily be produced. In addition, where the transgene expresses β-galactosidase derived from Aspergillus niger, the enzyme is particularly useful for hydrolyzing lactose at an acidic pH (at pH 3-4). Accordingly, a sample of milk including this enzyme is particularly useful for reducing the lactose content of a second sample of milk by simply mixing the two milk samples together. ' .

Where the heterologous enzyme is an aspartic protease, the milk is particularly useful for producing cheese. Such proteases decrease the time required for milk to be clotted by rennet. Aspartic proteases can also increase the yield of cheese. The expression of a bovine β-casein in milk can also improve cheese yields. In addition, the production of bovine β-casein or other heterologous proteins (e.g., lactoferrin or lysozyme) in milk can increase the nutritional value of the milk.

In additional embodiments of the methods of the invention, the promoter is a urinary endothelium-specific promoter, for example, a uroplakin promoter or a uromodulin promoter, or the promoter is a mammary gland- specific promoter, for example, a WAP (whey acidic protein) a casein promoter.

Dwarf goats are indigenous to India, Arabia, China, West Indies and

Africa (especially Nigeria, Ghana, and the Cameroons). Dwarf goats predominate in western Africa due to their natural resistance to the tse tse fly which destroys other goat types. This West African goat is also called the Fouta Djallon, Cameroon, Nigerian or tree goat.

The dwarf goats are homozygous for small size. At birth, the typical dwarf kid is less than half the size of a standard kid at birth. Dwarf goat birth weights average 1.4 kg for females and 1.6 kg for males while for standard Saanens the birth weight are 3.6 kg for females and 3.8 kg for males. Hybrid dwarf-standard kids are intermediate between parental phenotypes. Dwarf goats have a shorter generation time than other dairy ruminants (standard goats, sheep, cows) owing to their sexually precociousness. Dwarf goats of both sexes mature rapidly. Lactation can be induced hormonally, thus even further shortening the time required to obtain transgenic milk (five months for fetus gestation and two to four months for doe growth).

The lactational performance of transgenic Dwarf goats is sufficient to allow them to be a viable model for heterologous protein production. Typical lactation produces on average 1.0-1.5 L/day for 10 months, a remarkable amount considering the Dwarf goats' small body size. This lactation performance is thus sufficient to satisfy the requirement for gram quantities of protein.

Ebert et al. (Bio/technology 12:699, 1994) describe the induction of milk containing of human tissue plasminogen activator from the mammary gland of transgenic goats from a first generation transgenic male. Ebert et al. (supra) also describe a useful method for induction of lactation in males using a hormonal regime. This method permits early assessment of a transgene expressed in the mammary glands, even in males.

Keskintepe et al. describe a method for developing morulae in vitro from immature goat oocytes (Zygote 2:97, 1994). This method can be used to provide zygotes and embryos for gene manipulation, host embryos for chimera production, unfertilized recipient oocytes for use in nuclear transfer, and embryos and oocytes for use in other techniques.

Embryonic stems cells (ESC) of the invention are useful in the production of transgenic animals. They can be genetically transformed and then used to form chimeric embryos by blastocyst injection, morula injection, aggregation, or other techniques. ESC harboring the transgene are incorporated into the germ line and participate in the production of reproductive cells, the offspring produced by the chimeric animals will be transgenic. ESC have several advantages: 1 ) they permit increased efficiency of transgenic animal production; 2) they can be transformed in vitro; 3) they can be screened for the presence of the transgene (Robertson, Biology of Reproduction 44:238, 1991); and 4) they can be propagated so that one can generate many identical transgenic animals. The use of ESC makes it possible to replace an existing gene with a genetically altered gene by homologous recombination (Thomas et al., Cell 51 :501 , 1987). Pieper et al. (Nucleic Acids Res. 20:1259, 1992) describes methods for introducing a transgene into a murine zygote by homologous recombination.

The embryonic stem cell colonies of the invention are passaged every 7 to 14 days as desired. Colonies can be passage while small and undifferentiated. or can be allowed to almost reach confluency. Large colonies may have areas of differentiated and undifferentiated cells. Undifferentiated cells can be preferentially removed for continued passage. In goats, somatic cells do not go past 18 passages whereas stem cells can be passaged up to 40 or more times. The goat ES cells provided herein have been passaged at least 60 times but can be taken up to 150 times or more.

ESC can be frozen 10% glycerol and 90% ESCM. ESC lines can be restarted from frozen cells. Frozen cells are thawed rapidly, washed free of cryoprotectant and plated onto fresh feeder layers.

In one embodiment, established ESCs (after 1 passage) can be plated onto gelatin coated tissue culture plate instead of feeder cell layers. The ESC medium is supplemented with BRL cell conditioned medium (60% BRL conditioned MEM supplemented with 40% ESCM and 0.1 mM β- mercaptoethanol).

In accordance with the methods of the invention, high-passage gES cells did not require a feeder layer, a property also seen in high-passage mouse ES cells (see Amit et al., Biol. Repro. 70:837-845 (2004) as well as for human ES cells (see Ludwig et al., Nat. Meth., 3:637-646 (2006)), although such human cells appear to benefit from the presence of feeder cell layers to maintain an undifferentiated state (thereby raising the cost and introducing quality control issues for potential human clinical applications).

The ESC prepared according to the invention have a large nuclear to cytoplasmic ratio. At high cell numbers, the ESC grow in a flat monolayer with indistinct cellular edges. Colony edge is distinct and smooth. Cell size is less than or equal to 21 μm. When plated as single cells or in small clumps they form a small mound which will later expand to large flat colony as numbers increase. Undifferentiated cells are alkaline phosphate positive and form simple embryoid bodies spontaneously as cell numbers (colony size) increases. Some colonies may spontaneously differentiated into large flat

(trophectoderm-Iike) cells and/or with cells which have morphological characteristics of nerve cells and/or muscle cells.

Dwarf goats produced according to the invention, using embryonic stem cells provided herein, represent a useful animal model for human clinical studies (as opposed to mice, which are much smaller). For example, such dwarf goats can be used to study arthritis since a goat has a larger anatomy and is closer to humans in size. The transgenic goats produced using the stem cells of the invention can also be utilized in other processes such as production of milk and cheese. Goat stem cells are commonly characterized by the presence of up to 4 markers as stated herein.

One of the most important benefits of discoveries regarding the genetic or environmental causes of Parkinson's disease (PD) is the subsequent development of animal models wherein therapeutic and preventative interventions may be studied. The ability to study disease progression over long periods of time is particularly important for neurodegenerative diseases. Such diseases typically develop over extended periods of time in humans. We believe, therefore, that the transgenic goat is the preferred species for evaluation of neurodegenerative disease due to its longer lifespan (6-8 years) and the consistent availability of biological material for surgical applications. For example, goat embryonic stem cells could be differentiated to neuronal cells in vitro and, in turn, selective cells from these neurons could then be transplanted into animals and evaluated to determine their ability to differentiate into neural tissue in vivo.

Embryonic stem cells selected for the appropriate incorporation of a transgene are injected into a host embryo, preferably when the host embryo is at the morula or blastocyst stage, although injection can occur when the embryo is even younger. The ESC used are preferably from selected colonies which are separated into small clumps of cells (preferably five to fifteen cells) either by mechanical or enzymatic (pronase or trypsin) treatment. These cells are injected into the blastocoel of blastocyst staged embryos or under the zona into the mass of morula or younger stage host embryos. Alternately, zona free morula (or younger) embryos can be cultured with ESC separated by enzymatic treatment, allowing ESC to be incorporated into the embryo. Host embryos can be in vivo or in vitro produced, diploid or tetraploid.

Butyrylcholinesterase (BChE) has been shown to be an effective treatment against multiple LD50s of organophosphates. A prerequisite for such use of BChE is a prolonged circulatory half-life. A means of achieving plasma stability and longer half-life of recombinant BChE is to provide a recombinant^ produced BChE fused to human serum albumin (hSA). This fusion protein is believed to exhibit high plasma stability and an advantageous distribution in the body, and is expected to be either weakly or non- immunogenic for the organism in which it is used.

The BChE enzyme amino acid sequences and hSA amino acid sequences of the fusion protein may or may not be separated by linker amino acid sequences (e.g., a poly-glycine linker). Such linker amino acid sequences are often included to promote proper folding of the different domains of a fusion protein (e.g., hSA domain and BChE enzyme domain). [Huston et al.. PNAS (1988) 85:5879-5883; Syed et al., Blood (1997) 89:3243- 3252]. For example, hSA may be fused to either the N-terminus or the C- terminus of BChE. In preferred embodiments, the hSA moiety is fused to the C-terminal end of the BChE enzyme. This fusion is expected to provide a fusion protein that maintains BChE catalytic activity.

In accordance with the invention, a means of producing recombinant BChE with a glycosylatiσn profile that more closely resembles that of the native enzyme, the present invention is directed to transgenic animals that express both a BChE enzyme and one or more glycosyltransferases in their mammary glands and/or urinary endothelium, as well as cultured mammalian cells that express both a BChE enzyme and one or more glycosyltransferases. The presence of the glycosyltransferases in the intracellular secretory pathway of cells that are also expressing a secreted form of BChE catalyzes the transfer of glycan moieties to said BChE enzymes.

By way of non-limiting the example, where the transgene encodes and expresses BChE, the procedure involves introduction of an expression construct comprising a nucleic acid sequence encoding a glycosyltransferase enzyme operably linked to elements that allow expression of the glycosyltransferase enzyme in the tissue of interest. A second expression construct, for example, one of the BChE-encoding expression constructs described herein, is also introduced. Alternatively, the BChE enzyme and the glycosyltransferase may be encoded in a single bi-cistronic construct. An example of a bi-cistronic construct would be a construct which comprises a WAP promoter; a nucleic acid sequence which encodes both a BChE enzyme and a glycosyltransferase, in which an IRES (internal ribosomal entry site) is included between the sequence encoding the BChE enzyme and the sequence encoding the glycosyftransferase; and signal sequences to provide secretion of the BChE enzyme and the glycosyltransferase. This construct may be introduced into the genome of a stem cell by techniques well known in the art including microinjection, electroporation, and liposome-mediated transfection, calcium phosphate-mediated transfection, virus-mediated transfection, and nuclear transfer techniques.

The preferred glycosyltransferase enzymes for use in accordance with the present invention are sialyltransferases. Other enzymes that alter the glycosylation machinery whose production within a host cell may be desirable include fucosyltransferases, mannosyltransferases, acetylases, glucoronyitransferases, glucosylepimerases, galactosyltransferases, β- acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases, and sulfotransferases. For a description of such transferases see, for example;

Hennet. Cell MoI. Life Sci. (2002) 59:1081-1095; Harduin-Lepers, et al.

Biochimie (2001) 83:727-737; and Takashima, et al. J. Biol. Chem. (2002)

277:45719-45728.

In the event that independent transcripts are used to encode the BChE enzyme and the respective glycosyltransferses, it is preferred that different promoters are used to express the different transcripts. For example, if the nucleic acid sequence encoding the BChE enzyme is operably linked to a mammary gland-specific casein promoter, it is preferred that nucleic acid sequence encoding the glycosyltransferase is operably linked to a different mammary gland-specific promoter, such as a WAP promoter. Although it is preferred to use different promoters in this instance, the invention also encompasses use of the same promoter.

The recombinant DNA methods employed in practicing the present invention are standard procedures, well-known to those skilled in the art (as described, for example, in "Molecular Cloning: A Laboratory Manual." 2.sup.nd Edition. Sambrook, et al. Cold Spring Harbor Laboratory: 1989, "A Practical Guide to Molecular Cloning" Perbal: 1984, and "Current Protocols in Molecular Biology" Ausubel, et al., eds. John Wiley & Sons: 1989). These standard molecular biology techniques can be used to prepare the expression constructs of the invention. Each expression construct will additionally comprise a signal sequence to provide secretion of the translated recombinant BChE from the host cells of interest (e.g., mammary or uroepithelial cells, or mammalian cell culture). Such signal sequences are naturally present in genes whose protein products are normally secreted secreted. The signal sequences to be employed in the invention may be derived from a BChE gene, from a gene specifically expressed in the host cell of interest (e.g., casein or uroplakin gene), or from another gene whose protein product is known to be secreted (e.g., from human alkaline phosphatase, mellitin, the immunoglobulin light chain protein lgκ, and CD33); or may be synthetically derived.

The BChE-encoding nucleic acid sequences of interest may be modified in their 5¹ or 3' untranslated regions (UTRs), and/or in regions coding for the N-terminus of the BChE enzyme so as to preferentially improve expression. Sequences within the BChE-encoding nucleic acid sequence may be deleted or mutated so as to increase secretion and/or avoid retention of the BChE enzyme product within the cell, as regulated, for example, by the presence of endoplasmic reticulum retention signals or other sorting inhibitory signals.

In addition, the expression constructs may contain appropriate sequences located 5' and/or 3¹ of the BChE-encoding nucleic acid sequences that will provide enhanced integration rates in transduced host cells [e.g., ITR sequences as per Lebkowski, et al. MoI. Cell. Biol. (1988) 8:3988-3996]. Furthermore, the expression construct may contain nucleic acid sequences that possess chromatin opening or insulator activity and thereby confer reproducible activation of tissue-specific expression of a linked transgene. Such sequences include Matrix Attachment Regions (MARs) [McKnight, et al. MoI Reprod Dev (1996) 44(2): 179-184 and McKnight, et al. Proc Natl Acad Sci USA (1992) 89:6943-6947]. See also Ellis, et al., PCT publication No.: WO95/33841 and Chung and Felsenfield, PCT publication No.: WO96/04390. In addition to the above-recited uses of ES cells in studying neurodegenerative diseases, the methodology and products presented herein also provide for the production of animal models for the study of disease, such as diseases of the nervous system, for example, diseases of the central nervous system. In one non-limiting embodiment, there is provided a transgenic goat whose nervous system produces a transgenic protein. Such protein may be a type of cell-surface marker, or a receptor protein, or an internal protein, such as one involved in impulse transmission.

In one embodiment, the transgenic animal, such as a transgenic goat, expresses one or more transgenes that direct development of neural stem cells for production of neurons, astrocytes and oligodendrocytes. Such neural stem cells are characterized by their ability to self-renew, the ability to differentiate into multiple phenotypic lineages and the presence of markers such as nestin and vimentin (characteristic of undifferentiated neural stem cells but not of proliferating neural stem cells).

In another embodiment, there is provided a transgenic goat, derived from ES cells, whose nervous system, or a part of whose nervous system, produces cells that over-express, or under-express, a selected protein molecule, for example, a receptor. In so doing, the role of said receptor can be better characterized and the effects of agents that bind to said receptor can be better delineated. Alternatively, a mutated, aberrant, or less-active or inactive receptor protein can likewise be produced by a transgene expressed specifically in neural cells. Such transgene can be controlled by promoters selective for neural cell expression.

In like manner, other proteins specifically utilized in neural cells can be over-produced, under-produced, or produced in mutated form in the transgenic goat of the invention. Because goats are close to humans in size and weight, the results of such studies are more reliably applied to disease conditions in humans. Other types of proteins that may be differentially expressed in neural cells for the purpose of studying their effects are proteins that promote neural stem cells growth, engraftment and differentiation. For example, stem cell engraftment can be followed using markers like BrdU (bromodeoxyuridine), N- CAM and Ki-67. The differentiation of stem cells into other types of neural cells can also be followed by suitable markers such as neurofilament 68kD or 7OkD or 20OkD, β-tubulin ill, GABA, NeuN, MAP 2a + 2b, DDC, synaptophysin, TH, and NSE (for neurons), A2B5, CNPase (2'3'-cyclic nucleotide 3'-phosphohydrolase), O1 , MBP, NG2, 04 and galactocerebroside (for oligodendrocytes), and S100 and GFAP (for astrocytes). While such markers are generally useful in mammals such as humans and mice, their relative expression in goat stem cells may vary and different such markers will be of use for different types of applications (i.e., dependent on the animal model to be produced).

Among the proteins that might be studied are those that facilitate engraftment of neural stem cells or the differentiation of stem cells into neurons, oligodendrocytes and astrocytes, as well as other types of cells. For example, neurotrophins (which include such proteins as Nerve Growth Factor (NGF)) are molecules that promote brain development and wiring, such as for memory and learning. The ability to produce animal models that over-express, or under express, such proteins, especially in selected areas of the brain, can provide enormous insight into brain development and function. Due to the trophic (i.e., ability to promote survival) and their tropic (i.e., ability to promote axonic growth) properties, their role in neural diseases such as Alzheimer's Disease and Parkinson's Disease may be better understood and aid in the finding of treatments for such maladies. Neurotrophins are known to bind to specific tyrosine kinase (trk) receptors in the brain (for example, in neurons trk A is the main receptor for NGF). Thus, the effects of various receptors for neurotrophins can be studied in transgenic goats the over-produce or underproduce such receptors, or that produce a mutated receptor (i.e., one having a selected mutation). For nuclear transfer nuclear donor source is a transgenic stem cell, preferably embryonic and preferably from a dwarf goat. The cytoplast/host source can be any goat oocyte, in vitro or in vivo matured, and is commonly enucleated. The host oocyte is enucleated (metaphase Il chromosomes removed) either by microsurgical or by centrifugation methods and resulting host cytoplast is activated by any of several means (e.g., cold shock, electrical pulse, calcium ionophore— DMAP treatment, ethanol, etc.) prior to or post nuclear transfer (depending on the cell cycle stage of the donor nucleus). Commonly, the ES cell-cytoplast is activated post transfer and fusion. The donor nuclei are obtained by either mechanical or enzymatic (for example, trypsin, protease) separation of the donor embryo or cell line. In one embodiment, the individual ES cells (or karyoplasts) are transferred to the enucleated oocyte (the cytoplast) under the surrounding zona pellucida such that there is contact between the plasma membranes of the karyoplast and cytoplast. The karyoplast and cytoplast are fused by methods well-known in the art, such as electrofusion, polyethylene glycol (PEG), fusogenic proteins, viruses (e.g., sendai virus), and the like. The new zygote is subsequently cultured to an appropriate stage for transfer to a recipient animal or frozen storage.

An alternative method to karyoplast/cytoplast fusion is that the donor nucleus can be injected directly into the ooplasm of the enucleated oocyte (Collas et al., Molecular Reproduction and Development 38:264 (1994)).

The zygotes produced by nuclear transfer techniques can also be combined with a host embryo (in the manner described above) to produce chimeras. Prather et al. (U.S. Pat. No. 4,994,384) and Massey (U.S. Pat. No. 5,057,420) describe nuclear transfer methods. Tatham et al. (Biology of Reproduction 53:1088, 1995) describe additional nuclear transplantation methods.

The transgenic dwarf goats produced by the methods of the invention are used to produce human proteins, such as therapeutic proteins (e.g., human growth hormone) and veterinary therapeutic proteins (e.g., IL-6) in the milk or urine of the non-human mammal, such as a dwarf goat. Production of the heterologous protein in a mammal facilitates post-translational modification of the protein and obviates expensive cell culture media used in in vitro methods of protein production. The invention also offers the advantage that the heterologous protein can be produced in large quantities. In particular, transgenic goats can also be used to alter the characteristics of milk.

Of course, transgenic goats can be used for many of the same purposes for which other transgenic animals have been used. Examples of such uses are found in the following references: Sarvetnick et al. (PCT Application No. PCT/US94/04708); Bjursell et al. (PCT Application No. PCT/SE93/00515); Lonberg (PCT Application No. PCT/US94/04580); and Abraham et al. (PCT Applicaton No. PCT/GB94/00569), which list is by no means exclusive.

By way of non-limiting example, expression of an appropriate transgene can cause alterations in the protein, lipid, or carbohydrate content of the milk. Useful milk products, such as those having a reduced lactose content, can readily be produced. In addition, where the transgene expresses β-galactosidase derived from Aspergillus niger, the enzyme is particularly useful for hydrolyzing lactose at an acidic pH (at pH 3-4). Accordingly, a sample of milk including this enzyme is particularly useful for reducing the lactose content of a second sample of milk by simply mixing the two milk samples together.

Where the heterologous enzyme is an aspartic protease, the milk is particularly useful for producing cheese. Such proteases decrease the time required for milk to be clotted by rennet. Aspartic proteases can also increase the yield of cheese. The expression of a bovine β-casein in milk can also improve cheese yields. In addition, the production of bovine β-casein or other heterologous proteins (e.g., lactoferrin or lysozyme) in milk can increase the nutritional value of the milk. In particular, the heterologous protein may be butyrylcholinesterase, preferably human BChE, and may even be fused to an additional molecule, such as human serum albumin.

A large body of literature has grown describing genes and expression control regions useful in the construction of transgenes in a variety of livestock. A non-exhaustive list includes: Groenen et al. (Livestock Production Science 38:61, 1994); Wilmut et al. (Experientia 47:905, 1991); Pursel et al. (J. Animal Sci. 71(suppl. 3):10, 1993); Clark et al. (U.S. Pat. No. 5,322,775); and Bleck et al. (PCT Application No. PCT/US92/06549). Expression constructs and genes used in livestock other than goats can, if required, be adapted for use in goats. Hurwitz et al. (PCT Application No. PCT/US/06300) describes expression constructs suitable for expression of a heterologous protein in the milk of a goat.

Preferably, any genetic construct (i.e., plasmid) encoding a desired transgene, such as human BChE, also includes a transcription termination region. Useful termination regions include a polyadenylation signal and the 3¹- end of the gene from which the promoter region of the genetic construct was derived. Other useful transcription termination regions include termination regions which are known to affect mRNA stability, such as those derived from the bovine growth hormone gene, globin genes, the SV40 early region or milk protein genes.

Optionally, the linear or circular genetic construct includes an intron which can increase the level of expression of the heterologous gene. Generally, the intron should be placed between the transcription initiation site and the translational start codon; 3¹ of the translational stop codon; or within the coding region of the gene encoding the heterologous protein. The intron should include a 5' splice site (i.e., a donor site), a 3¹ splice site (i.e., an acceptor site), and preferably includes at least 100 nucleotides between the two sites. Particularly useful introns are those which are naturally found in genes of ruminants (e.g., genes encoding caseins).

Practically any heterologous protein can be produced in a transgenic dwarf goat. Particularly useful heterologous proteins include those which are of therapeutic value to humans or animals (e.g., htPA, hGH, and IL-6). Other particularly useful proteins include those which increase the nutritional value of the milk (e.g., β-casein and lactoferrin). Many genes encoding these and other useful proteins have been identified and cloned, allowing them to be readily subcloned for use in the production of transgenic dwarf goats.

Other particularly useful heterologous proteins include those which are valuable in food science. Among the useful proteins are those which possess an enzymatic activity directed toward a component of milk; such enzymes can be used to alter the lipid, protein, or carbohydrate content of the milk. For example, β-galactosidase can be produced with the invention to produce milk with a reduced lactose level. Genes encoding β-galactosidase can be derived from any of a number of organisms, including Aspergillus niger, (Kumar et al., 1992, Bio/technology 10:82); Homo sapiens (Oshima et al., 1988, Biochem. Biophys. Res. Comm. 157:238); Kluyveromyces lactis (U.S. Pat. No. 5,047,340; Sreekrishna and Dickson, 1985, Proc. Natl. Acad Sci. 82:7909; and Poch et al., 1992, Gene 118:55); Lactobacillus bulgaricus (Schmidt et al., 1989, J. Bacteriology 171 :625).

Useful proteins include: cytokines, aspartic proteases, lysozyme, stearyl-CoA desaturase, lipases, galactosyltransferase, blood clotting proteins, protein C, α1 -antitrypsin, urokinase plasminogen activator, human serum albumin, cystic fibrosis transmembrane conductance regulator, gamma-interferon, human CD4, growth factors, peptide hormones, oncoproteins, tumor suppressor proteins, milk proteins, hormone receptors, translation factors, transcription factors, acetylcholinesterases and butyrylchotinesterases. If desired, the gene encoding the heterologous protein can be mutated. Particularly useful mutations include mutations in the 5'- or 3'-untranslated regions of the gene, because such mutations may improve expression of the gene encoding the heterologous protein. Other useful mutations or deletions are those which increase secretion of the protein from the cell or inhibit retention of the protein inside the cell. For example, sequences encoding endoplasmic reticulum retention signals or other sorting inhibitory signals are preferably deleted from the genetic construct or mutated to be non-functional. In addition, truncated versions of naturally-occurring proteins can be used in the invention, provided that the truncated protein possesses a useful biological activity.

Each heterologous protein produced according to the invention should be bonded to a signal peptide if the protein is to be secreted from the mammary epithelial cell. The signal peptide can be a naturally-occurring component of the heterologous protein (e.g., the signal peptide of human placental β-galactosidase). Where the heterologous protein is not naturally a secreted protein, if secretion is desired, the genetic construct should be assembled such that a signal peptide is bonded to the heterologous protein so that the signal peptide directs secretion of the protein from the cell. Useful signal peptides can be derived from genes such as casein genes, the gene for human alkaline phosphatase, or the gene for melittin.

EXAMPLE 1

To derive gES cells for characterization, goat compact morulae and blastocysts were collected from superovulated adult Saanen crossbreed donors 7 days after insemination with a fertile male. Formation of the ICM disc structures, seen on floating day 7 in in vivo derived blastocysts cultured for 2 to 5 days in vitro (see Fig. 1A). This disc was isolated by cutting the trophectoderm. Embryos were collected by uterine flushing using a 12Fr. Foley catheter by means of a laparoscopically-assisted midventral laparotomy under general anesthesia.

To derive the gES cells to be characterized, a total of 58 in vivo produced embryos were recovered, of which 28 in vivo derived blastocyst- stage embryos were cultured on goat fetal fibroblast feeder layer (inactivated by mitomycin C) in a medium of DMEM containing 0.1 Mm I²- mercaptoethanol, 0.1 Mm MEM non-essential amino acids, 200 Mm L- Glutamine and 10% fetal calf serum (FCS). Using goat feeder cells eliminates any species cross-contamination and goat fetal fibroblasts are easy to expand so that a single cell line can be used for a sufficient period of time that variability between feeder cell lines is reduced. Caprine fetal fibroblast cell lines used as feeder lines were derived from 35-day fetuses that were surgically recovered. Single-cell suspensions were prepared by mincing fetal tissue in PBS and then cultured in M199H medium (Invitrogen, Burlington, Canada) containing 10% goat serum. After 4 days in culture, cells were harvested from confluent monolayers by trypsinization and gentle pipetting and arrested with mitomycin C (10 μg/ml) for 2 hours. Following 3 days in culture, 25 of the 28 embryos hatched. Of the 28, ten embryos had attached to the feeder layer, 9 had degenerated whereas the other 9 were floating in the medium and expanding in size. After 5 to 7 days in culture, 4 of the 10 attached embryos appeared with a prominent ICM disc surrounded by trophoectoderm cells (see Fig, 1B).

Inner cell mass cells (4/10) and embryonic discs (3/6) were isolated mechanically (Fig. 1C) and cultured on goat feeder cells in DMEM containing 10 ng/ml (1 ,000 U/ml) human leukemia inhibitory factor (hLIF) and 10% FCS. The ICM and embryonic disc outgrew into colonies on day 4 post-culture (Fig. 1 D). Compact colonies of cells from these outgrowths were isolated mechanically and passed onto fresh goat feeder cells every 4 to 5 days with the addition of 10 ng/ml hLIF into the culture medium. Colonies that formed from these outgrowths of either ICM or embryonic discs were cultured on feeder cells up to 20 passages. Overall success of gES derivation was as follows: total embryos 28, of these, hatched 25 (89%), attached 10/25 (56%), inner cell mass 4/10 (40%), embryonic disc 3/6 (50%) and ES cell (7/7 (100%).

Established colonies at passage 6 were tested for immunoreactivity against alkaline phosphatase (ALP) and Oct-4 using standard protocols. Characterization studies showed that these cell lines met all of the essential criteria that are generally used to define embryonic stem cells: (1) long-time serial culture without differentiation, (2) expression of specific stem cell markers, and (3) evidence of pluripotency by differentiation to somatic cells.

Thus, embryonic stem cell-like colonies (57%) were established from both ICM and embryonic discs cultured on goat fetal fibroblast feeder cells.

Colonies forming from these outgrowths (50%) of either ICM or embryonic disc stained positive for both AP and Oct-4 (See Figure 3). Embryoid bodies formed from colonies of either ICM or embryonic disc in suspension (DMEM containing 10% FCS with no hLIF). Two cell lines (one from ICM and one from embryonic disc) were then successfully maintained through multiple passages (at least 8) and were successfully cryopreserved. These gESC-like lines were then available for production of transgenic animals.

EXAMPLE 2

Passaging of Cultured gES Cells

One gES cell line (dubbed 15AA (see Figure 6) and confirmed by karyotype and PCR to be 60, XY) was cultured through passage 102, some

10 times more passages than is possible for goat fetal fibroblasts. This appears to be the first instance of continuous culture of a gES cell line for a period exceeding 1 year. After passage 43 (in medium recited in Example 1), the gES cells were cultured without the use of goat fetal fibroblast feeder cells but in the presence of hLIF. Harvesting of the cells was carried out using trypsin but without differentiation. All 7 gES lines (see Example 1) were cryopreserved at different passage numbers, with one line (15AA) maintained in cultured for about 2 years. The doubling time of late-passage gES cells without a feeder layer was 18 to 24 hours.

EXAMPLE 3 Expression of Characteristic Stem Cell Markers

Goat ES cells maintained in an undifferentiated state exhibited characteristic pluripotent cell markers. At the early passages (P < 35) gES cells were maintained in an undifferentiated state but required the presence of feeder cells and LIF. Using the LIF alone, without the goat feeder cells gave rise to differentiation, showing that the goat feeder cells used in these cultures efficiently Inhibited gES differentiation.

In accordance with the foregoing, every 4 to 5 days the non- differentiating goat ES cell colonies were pooled and dissociated by pipetting and transferred into a new dish with fresh goat feeder cells. Digestion with trypsin at early passages (P<35) promoted differentiation of the gES cells (e.g., to epithelial-tike cells - see Fig. 2B). The resulting data showed the similarities of gES cells with human and mouse ES cells where feeder cells and LIF are also needed for inhibition of differentiation at early passages and use of trypsin at early passages promotes ES cell differentiation.

Goat ES cells have a high nucleus to cytoplasm ratio so goat feeder cells were passaged on a new feeder layer at the same time as goat ES-like cells. The feeder cells did not form the ES cell-like colonies and did not survive after 4 weeks of culture, lmmunocytochemistry was used to characterize the expression of proteins known to be expressed by ES cells. Thus, the Pit-Oct-Unc (Oct-4) factor is known to be involved in the maintenance of undifferentiated ES cells, and their longevity so that the presence of Oct-4 and alkaline phosphatase (ALP) is useful in characterizing stem cells. For example, stage specific embryonic antigen (SSEA-1) is expressed in mouse ES cells but absent from human while SSEA-4 is present in human ES cells but absent from mouse, lmmunocytochemostry of the gES cells produced herein showed that these cells produced Oct-4, ALP, SSEA-1 and SSEA-4 markers (see Figure 3).

EXAMPLE 4 In Vitro Differentiation of gES cells

Goat ES cells produced in Example 1 were cultured a suspension in DMEM with 15% FBS with LIF to promote the formation of embryoid bodies (see Fig. 2A and 2C). EBs floated in suspension and culture without hLIF gave rise to frequent spontaneous differentiation into neuron-like cells (see Fig. 2F). Selection of EBs having a dendrite sphere-like morphology (Fig. 2D) promoted gES differentiation into neuronal cells that attached to cell culture plates and further differentiated, lmmunocytochemistry using an anti-nestin polyclonal antibody (from abCam Inc., Cambridge, MA, USA) demonstrated nestin expression in differentiated gES cells (Fig. 2E). Nestin is a large intermediate filament protein of class Type Vl that is expressed during neuronal development in primitive neuroepithelium. Cells with neuron-like morphology were observed after 5 to 8 days in culture (see Fig. 2F).

EXAMPLE 5 Goat ESC Transfection with GFP

Goat ES cells were obtained that have GFP (Green Fluorescence

Protein) and hBChE (human burtyrylcholinesterase) integrated into their genome. The GFP positive cells are then available to generate chimera or transgenic animals by nuclear transfer and thereby demonstrate embryonic stem cell plufipotency (if the GFP cells contribute to all cell lineages and germ cells).

For these experiments, a GFP plasmid was used at 0.4 μg/uL and monomeric BChE with ineobcNMBChE at 0.175 μg/uL, Goat transgenic lines were generated through lipofection (lipid-mediated gene transfer). The CEeGFP plasmid (obtained from Dr. T. Takada, National Children's Medical Research Center, Tokyo, Japan) contained the enhanced, humanized version of the green fluorescent protein reporter gene driven by the human elongation factor-la promoter and cytomegalovirus enhancer, and a neomycin selection marker under the control of the SV40 promoter. This plasmid was delivered into the cells (from line 15AA, see above) using lipofectamine (Gibco) according to the manufacturer's instructions. A number of stable clones were generated by selection under G418 for 20 days and assessed for expression of the reporter Reporter gene by visualization of the fluorescent signal under blue light (Zeiss Filter Set 09; Carl Zeiss Canada Ltd., North York, ON₁ Canada). Results are shown in Fig. 4.

For these experiments, cells were split 24 hours in advance so as to be approximately 70% confluent at the time of transfection. For ES cells in 6 well dishes, about 1.5x10⁶ to 2.0x10⁶ cells per well gave the required confluency.

A kill curve was performed the first time an antibiotic was used for selection.

DNA was prepared in advance of the experiments.

In each case, a negative control (gES cells treated by plasmid with no heterologous DNA or transgene) was used. Cells underwent all conditions of transfection, i.e., they were treated with Enhancer, EC buffer, and Effectene Reagent) . Enough volume of DNA (in μl_s) was added to obtain 0.4 μg/well. The DNA was added along with 100μL of EC buffer, and 3.2 μL of Enhancer to a screw cap tube, then mixed and allowed to stand 5 mins. Effectene reagent (from Qiagen Effectene Transfection Reagent kit (cat #301425)) was added, mixed, and allowed to stand 15 minutes more. During this time, the cells were washed 3 times with PBS and medium added onto the cell layer (1600 μL/well for 6 well plate). After 15 minute incubation, about 6O0μL of medium was added per well for the 6 well plate and the reaction mix in screw cap tubes was transferred to an appropriately labeled well in a drop-wise manner. The tubes were incubated overnight at 37°C.

After 24 hours, the medium was changed and cells split into 100mm dishes that had been coated with gelatin. Each well of a six well plate was split into two 100mm dishes, resulting in about 40,000 cells per 100mm dish. Selection was made by adding G418 Geneticin from Invitrogen (cat # 10131- 035) about 24 hours after splitting the cells and the medium changed every 2 days after selection was begun.

Clone Selection

In a hood, cylinders were placed small side down into vacuum grease

(before picking clones vacuum grease was autoclaved, along with forceps, and cloning cylinders) and with forceps pressed down evenly and gently on top. Under a microscope, clones were selected that appeared healthy (Round shape, small size (20-30μm), packed and colonies have more than 50 cells) Round shape, small size (20-30μm), packed and colonies have more than 50 cells.

Healthy colonies (meaning round in shape and of small size (20-30 μm), packed and with more than 50 cells) contain cells which are small, round, refractive and undifferentiated. Colonies should be isolated and contain at least 100 cells, and that were not too close to other colonies. For GFP transfected cells a fluorescent microscope was used. The best clones had about 100 cells. Plates were washed with 1x PBS and the cloning cylinders placed overtop of the clones (vacuum grease side down) and pressed down gently with thumb. Once all the clones on the plate had a cloning cylinder, 50μL of trypsin was added to each cylinder and placed on a plate heater under a hood for 5 minutes. After five minutes, 5OuL of media was placed into each cylinder (pipetted straight up and down several times, being careful not to touch the sides of the cloning cylinder) and medium was then transferred with the selected cells to a well already containing 1mL of stem cell media (DMEM (Gibco 10829-018), 20% fetal bovine serum (Gibco 10099-141), 2 mM L-glutamine (Sigma G6392), MEM non-essential amino acids (Sigma M7145), 1% MEM vitamins (Sigma M6895), 35 μg/mL gentamicin (Gibco 15710-072), 93 nM β-mercaptoethanol (Sigma 7522) and 10⁴ U/mL LIF (Chemicon International LIF-1010)) in a 48 well dish. Media also contained the selection moiety (G418). This was repeated for each plate. The cloning cylinders were removed and placed in a 5OmL falcon tube with 70% ethanol. All clones need not be collected in a single day.

Clonal Analysis

Once clones reached confluency in the 48 well dish, they were transferred to a 6 well dish and the cells washed twice with PBS. Two hundred microliters of trypsin was added and the cells incubated at 37⁰C for 5 minutes. Another 200 μL of medium was added and the cells transferred from each clone to two wells of a 6 well dish, keeping the clones separate. The cells were then inclubated in 2 mL of stem cell medium a medium of DMEM containing 0.1 Mm l²-mercaptoethanol, 0.1 Mm MEM non-essential amino acids, 200 Mm L-Glutamine and 10% fetal calf serum (FCS) (containing the appropriate selection moiety) DMEM (Gibco 10829-018), 20% fetal bovine serum (Gibco 10099-141), 2 mM L-glutamine (Sigma G6392), MEM non- essential amino acids (Sigma M7145), 1% MEM vitamins (Sigma M6895), 35 μg/mL gentamicin (Gibco 15710-072), 93 nM β-mercaptoethanol (Sigma 7522) and 10⁴ U/mL LIF (Chemicon International LIF-1010) Geneticin (G418) from Invitrogen (cat # 10131-035).

Once the cells reached confluency in the 6 well dishes, the cells in one well were frozen for future use and the cells in the second well used for analysis. Several methods are available to determine if the transfection was successful, including PCR and southern blot. If GFP was transfected, expression were confirmed using a fluorescent microscope.

GFP transfection was successful and GFP expression in the ES cells was confirmed by visualization under a fluorescent microscope.

Further, a most important difference between gESCs and somatic cells are demonstrated after transfection. Goat somatic cell division continues for 20-30 cell cycles while the morphology of cells appears damaged, with cells falling into senescence after several passages. In contrast, as shown in Figures 4 and 5, gESCs, following transfection, retain their pre-transfection morphology and continue dividing for over 100 cell cycles without entering a senescence stage.

EXAMPLE 6 Goat ESC Transfection with hBChE

Using a procedure similar to Example 5, the goat ES cell line 15AA was transfected with a construct encoding the monomeric form of human butyrylcholiπesterase (hBChE) (see, for example, Huang et al. US

2006/0253913 (9 November 2006) and Karatzas et al., US 2004/0016005 (22

January 2004)). A total of 19 clones were isolated and the results from testing

8 clones are shown in Fig. 6. All the clones were positive for monomeric BChE and the insulator region around the transgene. Amplification of an endogenous β-casein gene served as control for DNA quality. Importantly, after transfection and selection of positive clones, the morphology and doubling time of the gES cells did not change while fetal fibroblast cells transfected (as a control) underwent sensescence after several additional cycles post-transfection.

Claims

WHAT IS CLAIMED IS:

1. An isolated goat embryonic stem cell (gESC) having the capacity for germ-line transmission.

2. The isolated gESC of claimi , wherein said cell has said capacity when introduced into a goat embryo.

3. The isolated gESC of claim 1 , wherein said embryonic stem cell is derived from the inner cell mass of a goat.

4. The isolated gESC of claim 1 , wherein said embryonic stem cell is derived from the embryonic disc of a goat.

5. The isolated gESC of claim 1 , wherein said goat is a dwarf goat.

6. The isolated gESC of claim 1 , wherein said embryonic stem cell is positive for at least 4 ESC markers.

7. The isolated gESC of claim 6, wherein said markers are ALP, Oct-4,

SSEA-1 and SSEA-4.

8. The isolated gESC of claim 1 , wherein said embryonic stem cell comprises a transgene.

9. The isolated gESC of claim 8, wherein said embryonic stem cell comprises more than one transgene.

10. The isolated gESC of claim 8, wherein said transgene comprises a DNA sequence in operable linkage with a selected promoter to express a protein.

11. The isolated gESC of claim 10, wherein said selected promoter is a tissue-specific promoter.

12. The isolated gESC of claim 11 , wherein said selected promoter is a mammary gland-specific promoter.

13. The isolated gESC of claim 12, wherein the mammary gland- specific promoter is a WAP (whey acidic protein)

14. The isolated gESC of claim 12, wherein the mammary gland- specific promoter is a casein promoter.

15. The isolated gESC of claim 11 , wherein said tissue-specific promoter is a urinary endothelium-specific promoter.

16. The isolated gESC of claim 12, wherein the urinary endothelium- specific promoter is a uroplakin promoter.

17. The isolated gESC of claim 12, wherein the urinary endothelium- specific promoter is a uromodulin promoter.

18. The isolated gESC of claim 11 , wherein said selected promoter is a cytomegalovirus (CMV) promoter.

19. The isolated gESC of claim 10, wherein said protein is butyrylcholinesterase (BChE).

20. The isolated gESC of claim 19, wherein sard butyrylcholinesterase is human butyrylcholinesterase (hBChE).

21. The isolated gESC of claim 10, wherein said protein is a butyrylcholinesterase (BChE)-hurnan serum albumin (HSA) fusion protein.

22. The isolated gESC of claim 21, wherein said butyrylcholinesterase is human butyrylcholinesterase (hBChE).

23. A goat embryonic stem (gES) cell line that is capable of remaining undifferentiated through at least 30 passages.

24. The gES cell line of claim 23, wherein said cell line is capable of remaining undifferentiated through at least 50 passages.

25. The gES cell line of claim 23, wherein said cell line is capable of remaining undifferentiated through at least 70 passages.

26. The gES cell line of claim 23, wherein said cell line is capable of remaining undifferentiated through at least 100 passages.

27. The gES cell line of claim 25, wherein said cell line remains undifferentiated without the presence of feeder cells for passages greater than 50.

28. The gES cell line of claim 26, wherein said cell line remains undifferentiated without the presence of feeder cells for passages greater than 50.

29. A method for producing a goat, comprising: (a) introducing a nucleus from a goat embryonic stem cell into an enucleated goat oocyte to form a zygote,

(b) transplanting said zygote into a pseudopregnant goat, and

(c) allowing the resulting embryo to develop to term.

30. The method of claim 29, wherein said nucleus of step (a) is part of a gES cell.

31. A method for producing a transgenic non-human mammal whose somatic and germ cells contain a transgene, comprising:

(a) introducing a nucleus from a non-human mammalian embryonic stem cell into an enucleated oocyte of the same or different species of non- human mammal to form a zygote, wherein said embryonic stem cell nucleus contains a transgene comprising a DNA sequence or genetic construct encoding a heterologous protein and operably linked to a promoter,

(b) transplanting said zygote into a pseudopregnant non-human mammal of the same or different species, (c) allowing the resulting embryo to develop to term, and

(d) identifying at least one non-human mammal wherein expression of said transgene results in the production of a protein encoded by said DNA in a tissue of said non-human mammal.

32. The method of claim 31 , wherein said stem cell and said oocyte are from the same species of non-human mammal.

33. The method of claim 32, wherein the embryonic stem cell of step (a) is the goat embryonic stem cell of claim 2.

34. The method of claim 32, wherein said same species is goat.

35. The method of claim 34, wherein said pseudopregnant non-human mammal is a goat .

36. The method of claim 31 , wherein said nucleus is part of an embryonic stem cell that is introduced into said enucleated oocyte.

37. The method of claim 36, wherein said nucleus is part of an embryonic stem cell and is introduced into said enucleated oocyte by fusing said enucleated oocyte with said stem cell.

38. The method of claim 31 , wherein said heterologous protein is butyrylcholinesterase (BChE).

39. The method of claim 38, wherein said butyrylcholinesterase is human butyrylcholinesterase (hBChE).

40. The method of claim 31 , wherein said heterologous protein is a butyrylcholinesterase (BChE)-human serum albumin (HSA) fusion protein.

41. The method of claim 40, wherein said butyrylcholinesterase is human butyrylcholinesterase (hBChE).

42. The method of claim 31, wherein said promoter is a mammary gland- specific promoter and said tissue is mammary gland tissue.

43. The method of claim 42, wherein the mammary gland-specific promoter is a WAP (whey acidic protein)

44. The method of claim 42, wherein the mammary gland-specific promoter is a casein promoter.

45. The method of claim 42, wherein said heterologous protein is secreted in the milk of said non-human mammal.

46. The method of claim 45, wherein said non-human mammal is a goat.

47. The method of claim 31 , wherein said promoter is a urinary tissue- specific promoter.

48. The method of claim 47, wherein the urinary endothelium-specific promoter is a uroplakin promoter.

49. The method of. claim 47, wherein the urinary endothelium-specific promoter is a uromodulin promoter.

50. The method of claim 47, wherein said heterologous protein is produced in the urine of said non-human mammal.

51. The method of claim 47, wherein said non-human mammal is a goat.

52. A transgenic goat produced by the method of claim 35.

53. A transgenic goat produced by the method of claim 36.

54. A transgenic goat produced by the method of claim 46.

55. A transgenic goat produced by the method of claim 51.

56. The isolated goat embryonic stem cell of claim 11 , wherein said selected promoter is a neuronal-specific promoter.

57. An isolated neural stem cell derived from an embryonic stem cell of claim 1.

59. An isolated neuron derived from the neural stem cell of claim 57.