WO1999021415A9 - Nuclear transfer for production of transgenic animal embryo - Google Patents

Abstract

A method of generating a transgenic animal embryo which method includes providing a donor nucleus and a recipient cell; removing the nucleus for the recipient cell; introducing the donor nucleus into the enucleated cell to produce a couplet; and maintaining the couplet in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape. The method can be applied for porcine embryos and is exemplified with heterogenic expression of α1,3-galactosyl transferase.

Description

NUCLEAR TRANSFER FOR PRODUCTION OF TRANSGENIC ANIMAL EMBRYO

This invention relates to the generation of animals by nuclear transfer, including but not limited to the generation of transgenic animals.

Nuclear transfer is the replacement of the nucleus of one cell with that of another. The ability to produce live offspring by nuclear transfer is an objective which has been sought for some time by animal breeders. The ability to produce cloned offspring in such a manner would enable the production of large numbers of identical offspring and the ability to genetically modify and /or select cell populations of the required genotype (e.g., sex or transgenic) prior to embryo reconstruction.

Whilst nuclear transfer has been described in some animals, the procedures used are often inefficient and have not yet been successfully applied to many species. Also, there are no examples of gene activity being modified in a large animal using nuclear transfer. Applicants have discovered that, in relation to porcine embryos in particular, cloning techniques known in the prior art for ovine and bovine animals (see, for example, WO97/07669 and WO95/17500) cannot be applied to porcine animals.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

In a first aspect of the present invention, there is provided a method of generating an animal embryo which method includes providing a donor nucleus, and a recipient cell; removing the nucleus from the recipient cell; introducing the donor nucleus into the enucleated cell to produce a couplet; and maintaining the couplet in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape.

As used herein, a "couplet" is a complex comprising an enucleated recipient cell and a donor nucleus, wherein the donor nucleus is not completely within the plasma membrane of the recipient cell. In preferred embodiments, the recipient cell is an oocyte and the donor nucleus is positioned between the oocyte membrane and the zona pellucida. A "donor nucleus" is the genetic material of a donor cell, whether or not bounded by one or more membrane. Oocyte nuclei are preferably the metaphase plate of the oocyte. A "recipient cell" is a cell which obtains a donor nucleus.

Applicants have discovered that the number of viable embryos produced may be significantly increased by permitting the couplet to be maintained in a quiescent state for a period sufficient to allow the cell to recover a substantially normal, e.g., generally circular, shape.

Whilst we do not wish to be restricted by theory, it is postulated that the quiescent period permits the cell to return to a more normal state after which cell fusion may proceed more efficiently.

The couplet may be maintained in a suitable medium for a period of approximately 3 to 8 hours, preferably approximately 4.5 to 6 hours.

It is desirable, however, that the quiescent period end before any, or any substantive division, ensues. In a preferred embodiment of the present invention, the method may include the preliminary step of subjecting the recipient cell to an activation step; and subsequently removing the nucleus from the activated cell.

Applicants have found, in this preferred embodiment, improved results where a preliminary activation step is undertaken. Preferably the couplet is subsequently subjected to cell fusion.

Where the preferred preliminary activation step is not utilized the couplet may be subjected to a cell fusion/activation step. For example, where electrical pulses are utilized for cell fusion, the voltage may be selected to simultaneously initiate activation. In a further aspect of the present invention there is provided a method of generating a transgenic animal embryo said method including providing a donor nucleus which has been genetically modified to eliminate an undesirable activity or to provide for a desirable activity, and a recipient cell; transferring the donor nucleus to the recipient cell to produce a couplet; and generating a transgenic animal embryo from said couplet.

The animal embryo may be of any type, and includes bird, fish, reptile and mammalian (including ungulate and primate) embryos including human embryos, e.g., murine, bovine, ovine, or porcine embryos. Preferably, the animal embryo is a porcine embryo, bovine embryo, murine embryo, or human embryo.

Preferably the transfer step includes removing the nucleus from the recipient cell; introducing the donor nucleus into the enucleated cell to produce a couplet; and maintaining the couplet in a suitable medium for a period sufficient to allow the cytoplasm to recover a substantially normal shape.

The donor nucleus may be of any suitable type and from any suitable species. The donor nucleus may be contained in a karyoplast or cell. The donor nucleus may be of embryonic, embryonal tumor, foetal, or adult origin. Donor nuclei may be prepared by removing the nucleus and a portion of the cytoplasm and plasma membrane surrounding it from early pre-implantation stage embryos (for example, zygotes, 4- to 16- cell embryos) for example using microsurgery. When nuclei from more advanced embryonic cells are used, the whole blastomere may be transferred to the recipient cytoplasm. Embryonic or foetal fibroblasts may be used. Embryonic stem (ES) cells [isolated from inner cell mass (ICM) cells, embryonic disc (ED) cells or primordial germ cells (PGC)] may be used. A cell line derived from an embryonal tumor may be used (e.g., embryonal carcinoma (EC) or yolk sac tumor cells). Adult cells such as fibroblasts may also be used. In this case, the whole cell may be fused to the recipient cytoplasm. The recipient cell may be of any suitable type and from any suitable species. Cytoplasts may be prepared from in vivo or in vitro produced oocytes. Cytoplasts may be prepared from oocytes arrested in the second metaphase of meiotic maturation (Mil oocytes). Other sources of cytoplasts include zygotes, fertilized oocytes, and 2-cell blastomeres. Cytoplast preparation involves the removal of the nucleus in a process referred to as enucleation. The nucleus may be removed by microsurgery. This may involve the removal of pronuclei or metaphase plate and surrounding cytoplasm from zygotes or oocytes, for example, by aspiration or embryo bisection. Such manipulation may follow incubation of the zygotes or oocytes in a microfilament inhibitor, for example, cytochalasin B (Sigma Cell Culture,

Sigma- Aldrich Pty. Ltd.), that relaxes the cytoskeleton and allows the removal of a portion of membrane enclosed cytoplasm containing the pronuclei or metaphase plate. Alternatively, nonphysical approaches such as inactivation of the chromosomes by UV (chemical) or laser irradiation may be used.

In another aspect, the invention provides a method for generating an animal embryo, the method comprising providing a donor nucleus and a recipient cell; removing a nucleus from the recipient cell; maintaining the recipient cell in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape; and transferring the donor nucleus to the recipient cell by direct injection to produce a reconstituted cell.

The donor nucleus may be transferred to the recipient cell by any suitable method. Such methods include, but are not limited to, microsurgical injection, and cell fusion mediated by, for example, electrical pulses (electrofusion), chemical reagents such as polyethyleneglycol, or the use of inactivated virus such as Sendai virus.

Preferably the donor nucleus is introduced under the zona pellucida. In a further preferred aspect, the donor nucleus is introduced into the enucleated cell substantially immediately after enucleation.

More preferably, the nucleus is removed from the recipient cell via an incision site and the donor nucleus is introduced into the enucleated cell through the same incision site. Cytoplast volume may be increased by fusing together zona pellucida free cytoplasts before, after, or at approximately the same time as donor nucleus fusion.

An animal embryo may be generated from the reconstituted cell or couplet by any suitable method. Embryonic development may be initially in vitro and subsequently in a surrogate. Thus, the reconstituted cell or couplet may be initially cultured in vitro to produce an embryo and then the embryo may be transferred to a surrogate for subsequent development into an animal. In vitro culture of the reconstituted cell or couplet may be in any suitable medium.

Activation occurs during fertilization when the penetrating sperm triggers the resumption of meiosis. Activation is characterized by calcium oscillation, release of cortical granules, extrusion of the second polar body, pronuclear formation and, ultimately, cleavage. The reconstituted cell or couplet may be treated with, for example, ethanol, calcium ionophore, or electrical stimulation to induce activation. Activation may be performed prior to or after transfer of the donor nuclei.

In a preferred embodiment of this aspect of the present invention, the donor nucleus may be from an embryo that is itself the product of nuclear transplantation. This is known as serial nuclear transfer.

Serial nuclear transfer may improve the capacity of differentiated nuclei to direct normal development. Whilst applicant does not wish to be restricted by theory, serial nuclear transfer is postulated to improve the developmental capacity of transplanted nuclei by allowing specific molecular components in the oocyte to assist in chromatin remodelling that is essential for nuclear reprogramming. Serial nuclear transfer is not restricted to a singular event but may be initiated on more than one occasion to sequentially improve conditions for chromatin remodelling, nuclear reprogramming, and embryonic development. The donor nucleus and recipient cytoplasm which are used in the method of the present invention may be of any suitable origin. Preferably, they are of porcine bovine, murine, or human origin. More preferably, they are from a MHC (SLA) miniature swine because isogenic embryos and cell lines may be derived from such animals (see below). The method of the present invention may be used to generate transgenic animals. For example, a new gene may be expressed and/or an existing gene may be deleted in the transgenic animal. The addition of new genes is technically less demanding than the deletion of existing genes.

As used in this specification the term "transgenic", in relation to animals and all other species, should not be taken to be limited to referring to animals containing in their germ line one or more genes from another species, although many transgenic animals will contain such a gene or genes. Rather, the term refers more broadly to any animal whose germ line has been the subject of technical intervention by recombinant DNA technology. So, for example, an animal in whose germ line an endogenous gene has been deleted or modified (either by modifying the gene product or pattern of expression) is a transgenic animal for the purposes of this invention, as much as an animal to whose germ line an exogenous DNA sequence has been added.

The donor nucleus may be genetically modified by modifying, deleting, or adding one or more genes. The gene(s) to be modified, deleted, or added may be of any suitable type. In a particularly preferred embodiment of this aspect of the invention, the αl,3-galactosyltransferase gene may be modified in transgenic swine. This gene is non-functional in humans and so it is no longer expressed. It does, however, still function in swine where its role is to add a specific sugar configuration to protein backbones. This glycosylation pattern is highly antigenic and humans carry high levels of antibodies to it. Swine antigens to which humans react strongly are described as xenoantigens and the reactivity of humans towards swine antigens is described as xenoantigenicity. The net result is the immediate rejection of transplanted swine tissue in human recipients. Inactivation, through gene modification, of the swine α 1,3-galactosyltransferase activity may make swine organs, tissues, or cells less susceptible to immune recognition following transplantation of said organs, tissues, or cells into humans. The process of modifying a gene may involve the introduction of one or more mutations in both copies of the target gene. Suitable cells may take up the mutation(s) and then be used to generate an animal. One copy of the gene may be disrupted in the cell and the resultant heterozygous animals bred with each other until one with both copies of the gene mutated is found. Alternatively, both copies of the gene may be modified in vitro.

To target an endogenous gene rather than introduce random mutations, a DNA construct (transgene) including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the target gene except for the introduction of the one or more mutations may be used.

The targeting DNA may comprise a sequence in which the desired sequence modifications are flanked by DNA substantially isogenic with a corresponding target sequence in the genome to be modified. The substantially isogenic sequence is preferably at least about 97-98% identical with the corresponding target sequence (except for the desired sequence modifications), more preferably at least about 99.0-99.5% identical, most preferably about 99.6% to 99.9% identical. The targeting DNA and the target DNA preferably share stretches of DNA at least about 75 base pairs that are perfectly identical, more preferably at least about 150 base pairs that are perfectly identical, even more preferably at least about 500 base pairs that are perfectly identical. Accordingly, it is preferable to use targeting DNA derived from cells as closely related as possible to the cell being targeted; more preferably, the targeting DNA is derived from cells of the same haplotype as the cells being targeted. Most preferably, the targeting DNA is derived from cells of the same individual (or animal) as the cells being targeted. Preferably, the targeting DNA sequence comprises at least about 100-200 base pairs of substantially isogenic DNA, more preferably at least about 300-1000 base pairs of substantially isogenic DNA, even more preferably at least 1000-15000 base pairs of substantially isogenic DNA.

As used herein, the term isogenic or substantially isogenic DNA refers to DNA having a sequence that is identical with or nearly identical with a reference DNA sequence. Indication that two sequences are isogenic is that they will hybridize with each other under the most stringent hybridization conditions (see e.g., Sambrook J. et al., 1989); and will not exhibit sequence polymorphism (i.e., they will not have different sites for cleavage by restriction endonucleases). The term "substantially isogenic" refers to DNA that is at least about 97-99% identical with the reference DNA sequence, and preferably at least about 99.5-99.9% identical with the reference DNA sequence ,and in certain cases, 100% identical with the reference DNA sequence. Indications that two sequences are substantially isogenic is that they will still hybridize with each other under the most stringent conditions (see Sambrook, J. et al., 1989) and that they will only rarely exhibit restriction fragment length polymorphism (RFLP) or sequence polymorphism (relative to the number that would be statistically expected for sequences of their particular length which share at least about 97-98% sequence identity). In general, a targeting DNA sequence and a host cell sequence are compared over a window of at least about 75 consecutive nucleotides. DNA sequences compared between individuals of a highly inbred strain, such as the MHC inbred miniswine, are generally considered to be substantially isogenic even if detailed DNA sequence information is not available, if the sequence do not exhibit sequence polymorphisms by RFLP analysis. Thus, the donor nucleus may be genetically modified by modifying an endogenous gene in the donor nucleus. The endogenous gene may be modified by introducing into said donor nucleus a DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations, such that there is homologous recombination between the DNA construct and the endogenous gene.

In a further aspect of the present invention, there is provided a DNA construct for modifying an endogenous gene in an animal nucleus, said DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations.

In a preferred embodiment of this aspect of the invention, the endogenous gene is a swine, bovine, ovine, caprine, or murine gene, more preferably a swine αl,3galactosyltransf erase gene. In a particularly preferred embodiment of this aspect of the invention, the DNA construct is pGallaway, as hereinafter described or a functionally equivalent plasmid.

By a functionally equivalent plasmid, we mean a plasmid capable of performing substantially the same function in substantially the same manner as the plasmid in question. Without limiting the generality of the foregoing, other plasmid backbones are contemplated. Also, plasmids containing other portions of the swine αl,3-galactosyltransf erase gene and other mutations to that gene are contemplated.

The introduction of new genetic material and the subsequent selection of cells harboring the desired targeted integration requires expansion and clonal selection of each founder transgenic cell. A limitation to applying this process in nuclear transplantation programs is the number of cell divisions which the transfected cell must undergo to provide sufficient material for molecular analysis of each transgenic colony and subsequent supply of nuclei for transfer. The great majority of cells suitable for in vitro genetic modification and subsequent nuclear transfer have limited in vitro propagation capacity. It is therefore desirable to utilize transfection and selection systems which generate and/or identify correctly targeted clones at high efficiency and with limited requirement for in vitro propagation.

A particularly efficient approach to selecting for correctly targeted clones is to use IRES gene trap targeting vectors, as described in Australian Patent 678234, the entire disclosure of which is incorporated herein by reference. The IRES gene trap targeting vector may be selected from IRES-neo, IRES-lacZ, (TAA₃) IRES-lacZ, (TAA₃) IRES-lacZ lox neo-tk lox, (TAG₃) IRES-lacZ/mclneo, SA lacZ-IRES neo, SA (TAA₃) LRES-nuclear lacZ, SA (TAA₃) LRES-nuclear lacZ lox Gprt lox, IRES- βgeo, (TAA₃) IRES- βgeo, SA IRES- βgeo, SA Optimised IRES- βgeo, LRES-nuclear βgeo, SA LRES-nuclear βgeo, SA (TAA₃) LRES-nuclear βgeo, SA Optimised LRES-nuclear βgeo, IRES-zeo, SA IRES-zeo, IRES-hph, SA IRES-hph, IRES-hph-tk, IRES-bsd, SA IRES-bsd, LRES-puro. IRES gene trap targeting vectors provide a significant enhancement in gene targeting efficiency by eliminating a large proportion of random integration events. IRES gene trap targeting vectors rely upon functional integration into an actively transcribed gene (such as the target gene) for expression of the selectable marker. Random integrations into non-transcribed regions of the genome are not selected.

Accordingly, in a further aspect of the present invention there is provided a DNA construct for modifying an endogenous gene in an animal nucleus, said DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations, and a selectable marker, such that functional integration of said DNA construct into said endogenous gene results in expression of the selectable marker.

In a preferred embodiment of this aspect of the invention, the endogenous gene is a swine, bovine, ovine, caprine, or murine gene, more preferably a swine αl,3-galactosyltransf erase gene. In a particularly preferred embodiment of this aspect of the invention, the DNA construct is pBERT I, as hereinafter described or a functionally equivalent plasmid.

In a preferred embodiment, it may be desirable to remove the selectable marker cassette from the targeted locus to eliminate expression of the, e.g., antibiotic resistance gene. One approach is to flank the IRES selectable marker cassette with suitable DNA sequences which act as recombination sites following the addition of a suitable site-specific recombinase. One example of a suitable recombinase site is the lox site which is specific for the Cre recombinase protein. Another example of a suitable recombinase is the FLP/FRT recombinase system (O'Gorman, S. et al., 1991).

Accordingly, in a further aspect of the present invention there is provided a DNA construct for modifying an endogenous gene in an animal nucleus, said

DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations a selectable marker, and recombination sites flanking said selectable marker, such that functional integration of said DNA construct into said endogenous gene results in expression of the selectable marker and addition of a recombinase results in removal of the selectable marker by recombination at the recombination sites.

In a preferred embodiment of this aspect of the invention, the endogenous gene is a swine or murine, more preferably a swine α 1,3-galactosyltransferase gene. In a particularly preferred embodiment of this aspect of the invention, the DNA construct is pBERT II, as hereinafter described or a functionally equivalent plasmid.

High efficiency gene targeting and selection has a significant advantage in that suitably stringent selection systems, such as the IRES gene trap targeting vectors, can eliminate the need for biochemical analysis of clonal cell lines. In this instance, individual nuclei from a pool of uncharacterized transgenic cells should generate offspring of the desired phenotype at a ratio equivalent to the selected pool. The elimination of clonal selection may be particularly useful where only limited in vitro propagation is desirable or possible. One such instance includes the culture of embryonic nuclei for nuclear transfer. Embryonic nuclei are more efficient than latter stage somatic cells for generating live born offspring by nuclear transfer. However, totipotential embryonic cells can not be cultured for extended periods for any other species than mice. Nuclear recycling of embryonic nuclei (see following) provides an opportunity to maintain, expand, and genetically manipulate multipotential cells from animals in vitro.

The DNA constructs according to this aspect of the invention may be engineered in bacteria and then introduced into the cells. The transgenes may be introduced into the cells by any suitable method. Preferred methods include direct injection, electroporation, liposomes, or calcium phosphate precipitation. Direct injection is the preferred method for embryonic cells while electroporation is more suitable for embryonic fibroblast and embryonic stem cell cultures. Whilst applicant does not wish to be restricted by theory, it is thought that regions of substantially isogenic DNA either side of the mutation drag the transgene to the target site where it recombines and introduces the mutation. It is further thought that the main contributing factor for increasing the efficiency of introducing a specific mutation in a given gene is the degree of similarity between the target DNA and the introduced DNA. Thus, it is preferred that the DNA is isogenic (genetically identical) not allogenic (genetically dissimilar) at the genetic locus that is to be targeted.

In a further aspect of the present invention, there is provided an animal embryo or transgenic animal embryo produced by the methods of the present invention. Preferably, the animal embryo or transgenic animal embryo is a porcine, murine, ovine, bovine, caprine, or human embryo.

In a further aspect of the present invention, there is provided a reconstituted animal cell or modified reconstituted animal cell produced by the methods of the present invention. Preferably, the reconstituted animal cell or modified reconstituted animal cell is a porcine, murine, ovine, bovine, caprine, or human cell.

In a still further aspect of the present invention, there is provided an animal or transgenic animal produced by the methods of the present invention. Preferably, the animal or transgenic animal is a porcine, murine, ovine, bovine, caprine, or human animal, more preferably a MHC (SLA) miniature swine.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures:

Figure 1A shows an overview of a nuclear transfer procedure according to the present invention in which activation occurs prior to enucleation which is then followed by a quiescent period of, e.g., 6 hours, and then, when applicable, fusion.

Figure IB shows an overview of a nuclear transfer procedure according to the present invention in which recipient cells are enucleated, subjected to a quiescent period of, e.g., 6 hours, and then activation or combined fusion/ activation.

Figure 2 summarizes the steps taken for construction of the pGallaway targeting vector, deposited in American Type Culture Collection, ATCC Accession

No. . Figure 3 is a map of pBERT.

Figure 4 is a map of the linker referred to in Example 3.

Figure 5 shows the modifications to intron 8 and exon 9.

Figure 6 shows the genomic organization of the swine αl,3-galactosyltransf erase gene following a homologous recombination event between the chromosomal locus and pGallaway vector.

Figure 7 shows the strategy used to identify mutant transfectants.

Figure 8 shows a PCR analysis of the transfectants obtained in Example 3.

Figure 9 shows the results of Ase I digestion of mutant PCR products.

Figure 10 is a map of pBERT I. Figure 11 is a map of pBERT II.

Figure 12 shows transgenic animals generated by nuclear transfer.

Figure 13A shows an RT-PCR analysis of the transfectants obtained in Example 5.

Figure 13B shows the results of an RT-PCR analysis of the transfectants obtained in Example 5 after Ase I digestion.

Figure 14 shows a strategy developed for screening for targeted colonies referred to in Example 12.

EXAMPLE 1 Cytoplast Preparation Cytoplasts were prepared from either in vivo or in vitro produced oocytes collected from both outbred and MHC (SLA) inbred miniature swine. Whilst applicant does not wish to be restricted by theory, it is thought that the use of the MHC (SLA) inbred miniature swine oocytes as a source of cytoplasts will be beneficial in reducing mitochondrial DNA chimaerism and extraneous agents present in the cytoplast.

In vivo oocyte production

A number of systems have been developed to synchronize and superovulate swine to obtain a large number of oocytes required for cytoplast preparation. These systems can be used in both outbred and MHC (SLA) miniature swine. Synchronization of the estrous cycle was achieved by the following treatments: 1. administration of synthetic progesterone (e.g., Regumate, Altrenogest, Hoechst -Roussel); 2. prostaglandin or prostaglandin analogue injection of a pregnant swine; 3. removal of offspring at weaning. Natural cycling swine was also used. Superovulation with follicle stimulating hormone (FSH) was used to increase the number of oocytes obtained from each ovary. The FSH activity of pregnant mares serum gonadotrophin (PMSG, Folligen, Intervet (Aust) Pty. Ltd? or Pregnecol, Horizon Technology Pty. Ltd., 750-1500 IU) is preferred. Ovulation time was controlled with the injection of human chorionic gonadotrophin (hCG (Chorulon, Intervet (Aust) Pty. Ltd., 500 IU). Unfertilized metaphase II oocytes were surgically collected 2-16 hours after ovulation.

In vitro oocyte production Pigs

Ovaries were collected from slaughtered gilts and transported to the laboratory at 37°C in Saline (0.9% Sodium Chloride) (HSA, Baxter AHB1324) supplemented with 10 μl/ml penicillin-streptomycin solution (contains 5,000 iu/ml penicillin G sodium and 5,000 μg/ml streptomycin sulfate, Gibco BRL, Life Technologies Inc., US).

Oocyte cumulus complexes (OCC) were aspirated from follicles 2-6 mm in diameter. OCC with uniform cytoplasm and compact cumulus cell mass were collected, rinsed in Dulbecco's phosphate buffered saline (Gibco BRL) supplemented with 0.3% bovine serum albumin (ICN Biomedicals Inc.) and cultured in one of the following media:

Medium 1 Medium 199 (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.) Medium 2 Modified Whittens medium (Whitten, W.K. and Bigger, J.D., 1968, Funahashi, H. et al, 1994b, 1996) Medium 3 Modified GI medium

A summary of porcine oocytes matured in the various media is shown in Table 1.

TABLE 1 In Vitro maturation of porcine oocytes Medium No. of oocytes examined No. of oocytes with first polar body (%)

M199 352 220 (62.5)

Whittens 330 205 (62.1)

GI 383 150 (39.2)

Waymouth 275 71 (25.8)

OCC were cultured in microdrops or Nunc 4-well tissue culture plates of one of the above oocyte maturation medium or any other suitable medium overlaid with mineral oil (Sigma Chemical Company), at 38.5°C under 5% CO_z in air for 48-50 hours. Each medium was supplemented with 10% Estrus cow serum (Day 0 to 5 after standing heat) and 0.01 units/ml Follicle Stimulating Hormone (from porcine pituitary, Sigma-Aldrich Pty. Ltd.). The benefits of adding of Estrus cow serum is shown in Table 2.

TABLE 2 Effects of different sera on the in vitro maturation of porcine oocytes

Sera No. of oocytes examined No. of oocytes with first polar body (%)

ECS 1187 728 (61.3)

FBS 271 86 (31.7)

*ECS, Estrus Cow Serum; FBS, Fetal Bovine Serum

**Basic medium, TCM 199; Maturation time 48-52 hrs

Cattle

In vitro oocytes were obtained in an identical manner to porcine in vitro oocytes. Maturation was also in identical media, over a period of 22 hours.

Microsurgical Enucleation or Bisection of nuclear material

Recipient cytoplast were prepared by removal of metaphase chromosomes from mature metaphase LI (Mil) oocytes, or both pronuclei (PN) from in vivo fertilized oocytes for the first and second round of nuclear transfer respectively.

Enucleation by microsurgery

Prior to enucleation, the oocytes were denuded of cumulus cells by gentle pipetting after 2 minutes vortexing in 0.1% hyaluronidase (Sigma Cell Culture, 300 iu/ml in Hepes buffered M199 or H-Gl (or other short term handling medium) and incubated in the UV fluorescent dye Hoechst 33342 (Sigma Cell Culture, 5 μg/ml) for approximately 10 minutes at 38.5°C followed by washing in Hepes (Sigma Cell Culture or Gibco BRL) buffered M199, GI or other medium (listed above). The enucleation was performed in a 20 μl drop of Hepes buffered medium (M199 or GI) containing the cytoskeletal inhibitor, cytochalasin B (Sigma Cell Culture, 1.0-7.5 μg/ml). Using the micromanipulation pipettes oocytes were orientated so that the first polar body (PB) was clearly visible in the 12 o'clock position when the bevel of the enucleation pipette was upward facing. Once the oocyte was secured, the enucleation pipette was carefully inserted into the oocyte. The alignment of the holding and enucleation pipette with respect to the oocyte, zona pellucida and oolemma (cytoplasmic membrane) was important. Failure to have both pipettes and oocyte aligned resulted in lysis of the oocyte. Having penetrated the zona and oolemma, the enucleation pipette was positioned just below the first polar body. Gentle suction was applied into the enucleation pipette to remove a proportion (approximately 1/3 to 1/8) of the oocytes cytoplasm and the first polar body. Successful enucleation was confirmed by the detection of the metaphase plate in the removed cytoplasm and first polar body by exposing the enucleation pipette to UV light. Care was taken to avoid exposure of the enucleated oocyte to UV. In mouse experiments, the first polar body was rarely removed as it is undergoing degeneration at this particular time. This enucleation procedure was performed at room temperature.

If the polar body was not observed prior to enucleation the oocyte was exposed to UV (<15 seconds) to allow for visualization and orientation of the metaphase II plate. Following enucleation the oocyte and confirmation of enucleation was identical to the methods described above.

Cytoplast from the second round of nuclear transfer were prepared ^rom in vivo sourced zygotes. Donor female gilts or swine were synchronized and superovulated as described above. Females were mated with males twice, 24 hours and 32 hours after hCG (Chorulon, Intervet (Aust) Pty. Ltd.) injection. Zygotes could also produced by in vitro fertilization of in vitro matured oocytes. Fertilized oocytes have two pronuclei: male and female pronucleus (PN), which appear 4-8 hours after fertilization. The developing pronuclei are normally obscured by the high content of lipids present in swine embryos. Visualization of the PN was achieved by centrifugation (6-12 min., 8,000-15,000g). Centrifugation stratifies the lipids and allowed both PN to be easily removed by enucleation techniques. Cumulus cells still adhered to the zona pellucida were removed by incubation in hyaluronidase (300 iu/ml) for approximately five minutes.

Cytoplasts for the purpose of serial nuclear transfer were prepared from zygotes 44-50 hours after hCG (Chorulon, Intervet (Aust) Pty. Ltd.) administration. Fertilized oocytes were incubated in cytochalasin B (Sigma-Aldrich Pty. Ltd.). In a manner similar to that described for MIL oocyte enucleation, both PN were removed by microsurgery. No DNA staining was required to confirm the removal of PN since they were clearly visible in the enucleation pipette.

Enucleation by bisection

In vivo or in vitro matured oocytes were used for enucleation by bisection. The oocytes were denuded of cumulus cells by gentle pipetting or digestion in hyaluronidase (Sigma Cell Culture, 300 IU/ml in H-Gl medium or other short term handling medium) and stained in UV fluorescent DNA dye Hoechst 33342 (Sigma Cell Culture, 5 μg/ml) for approximately 10 minutes at 39°C. Metaphase LI oocytes were selected. The zone pellucida was partially dissolved by a short incubation in 0.1% pronase (Sigma Cell Culture) and removed by a gentle passage through a narrow pipette. The oocytes were incubated 15 min in H-Gl or other short term medium with cytochalasin B (Sigma Cell Culture) (1.0 - 7.5 μg/ml) and enucleated by bisection into two halves (Tarkowski, 1977). The nucleus was likely to be located in close proximity to the first polar body. Phytohemagglutinin was used to stick the polar body to the cytoplasm membrane. The enucleated halves, identified under UV illumination by the absence of chromatin stained with

Hoechst^~(Sigma Cell Culture), were washed and stored in H-Gl medium or other short term medium until used for fusion (46-50 hours after the onset of oocyte maturation).

EXAMPLE 2 Karyoplast preparation

Karyoplasts from the following sources were used: 4- to 16-cell embryos (embryonic nuclei), embryonic foetal, and adult (somatic) fibroblasts, embryonal tumour (Embryo Carcinoma) and embryonic stem cells (isolated from inner cell mass cells), embryonic disc cells, or primordial germ cells.

Karyoplast preparation from Preimplantation embryos.

Preimplantation embryos at 4 to 16-cell stage were collected from the reproductive tracts of superovulated and mated female miniswines. Superovulation was induced as described in Example 1. Foetuses for PGC isolation were collected from 25 to 35 day pregnant female miniswine following superovulation or natural cycling and mating (Example 1).

Four to 16-cell stage embryos were collected using standard surgical procedures 94 to 144 hours after hCG (Chorulon, Intervet (Aust) Pty. Ltd.) injection. Karyoplasts containing embryonic nuclei were obtained by removing a portion of the cytoplasm which contained the nucleus of the blastomere. This was performed by microsurgery in a similar manner to that described above for cytoplast preparation. The embryo was incubated in a microfilament inhibitor (e.g., cytochalasin B. (Sigma Cell Culture)) prior to the microsurgery, held in position by a holding pipette, the enucleation pipette was inserted between the blastomeres, and a portion of the cytoplasm containing the nucleus was removed. Karyoplasts were prepared from the blastomeres in the late G2 or M- phase of the cell cycle. The M- phase was induced by culturing embryos in a microtubule polymerization inhibitor, for example, nocodazole (Sigma Cell Culture, 1 μg/ml) (Kwon and Kono, 1996). Alternatively the whole blastomeres, synchronized in the G2 or M- phase were used as the source of the karyoplasts. The cell cycle consists of four phases: M (mitosis), GO, S (synthesis), and G2. Synchronization of cell cycles between donor nuclei and recipient cytoplasts at the time of transplantation strongly influenced the development of reconstituted eggs.

Karyoplast preparation from fibroblasts and embryonic cells. Foetuses were collected on day 25 to 35 of pregnancy from slaughtered female miniswine. The uterus was removed and foetuses isolated using sterile techniques. The foetuses were decapitated immediately after isolation and washed in cold PBS (Oxoid, Unipath Ltd., UK). A small tissue sample was taken from each foetus for genotyping (by using PCR). The fibroblast cultures were established from lung, muscle/skin (mesenchymal) tissues as follows. Other tissues may also be used. The tissue from individual foetuses was dissected, washed in PBS (Oxoid, Unipath Ltd.), disaggregated mechanically by mincing with scissors or/and enzymatically by incubating for 5 min at 37°C in 0.25% trypsin / 0.04% EDTA (Gibco BRL) in PBS (Oxoid, Unipath Ltd.). Disaggregated cells were washed in culture medium and plated onto gelatinized 10 cm Petrie dishes, at 10 - 10 cells per dish. This is passage 0 (PO). The medium was changed every 24 hours and the cells cultured until confluent (between 2 to 5 days). The cells were then expanded by trypsinization and passaging 10 - 10 cells onto a 10 cm gelatinized Petrie dish in culture medium. Aliquots of cells at passage 0, 1, 2 were stored frozen in liquid nitrogen. The culture medium was DMEM (Gibco BRL) supplemented with 10% FCS, 2 mM glutamine (Gibco BRL), 100 IU/ml penicillin (Gibco BRL), 100 μg streptomycin (Gibco BRL), 0.1 mM β mercaptoethanol (Gibco BRL). Other types of media or protein/ growth factors may also be used (e.g., DMEM (Gibco BRL): F12 (Gibco BRL)), if they are found to provide better culture conditions. The cells were karyotyped at each passage to confirm that they retained normal /diploid chromosome complement.

Other types of cells used as a karyoplast source include tissue from adults and embryonic stem cells isolated from ICM, ED, PGC, and EC.

PBS

Sterile solution of PBS (Oxoid, Unipath Ltd.), (Ca⁺⁺ and Mg⁺⁺ free) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin.

Culture media

Dulbecco's modified Eagle's medium (DMEM, (Gibco BRL)) supplemented with 10-20% FCS (CSL Limited, Aus.), 100 IU/ml penicillin (Gibco BRL), 100 μg/ml streptomycin (Gibco BRL), 2 mM glutamine (Gibco BRL), 0.1 mM β mercaptoethanol (Gibco BRL).

Gelatin

0.1% gelatin (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.) in water.

Trypsin

0.25% trypsin (Gibco BRL), 0.04% EDTA (Gibco BRL) in PBS (Oxoid, Unipath Ltd.), (without Pen/Strep).

Freezing

In 90% culture medium, 10% DMSO (Sigma Cell Culture). EXAMPLE 3 Isolation and genetic modification of bovine embryonic fibroblasts Isolation and propagation

A bovine foetus (CR length 7.5 cm) was collected from an abattoir -slaughtered cow. The foetus was washed in 3 changes of cold PBS containing 100 IU/ml penicillin, 100 μg streptomycin (PBS-P/S) eviscerated and washed again in 3 changes of cold PBS-P/S. Fibroblast cultures were established from superficial skin (BEF-S) and from muscle (mesenchymal; BEF-M) tissues as follows. The superficial skin was peeled off from hind and front legs and the muscle tissue was dissected from hind legs, washed in 2 changes of cold PBS-P/S, disaggregated mechanically by chopping with scalpel blades followed by enzymatic digestion. For enzymatic digestion tissues were incubated for 90 min (or up to 4 - 6 hr) at 4° C in 0.25% trypsin / 0.04% EDTA in PBS. After removing the excess of trypsin, tissues were incubated at 37 °C for 10 - 20 min., culture medium was added and cells disaggregated by vigorous pipetting. Large, undigested pieces of tissue were allowed to settle by sedimentation. Supernatant containing single cells was decanted and the cells plated at 3 x 10⁴ cells/cm² onto gelatinized (0.1% gelatin in water) Petrie dishes and cultured at 37 °C in an atmosphere of 5% CO_z in air. This was passage 0 (PO). The medium was changed every 24 hours and the cells cultured until confluent (between 2 to 5 days). The cells were then expanded by trypsinization and passaging 10⁵ - 10⁶ cells onto a 10 cm gelatinized Petrie dish in culture medium. Aliquots of cells at passage 0, 1, 2 were stored frozen in liquid nitrogen (in 10% DMSO, 90% culture medium or FCS). The culture medium was DMEM supplemented with 15% FCS, 2 mM glutamine, 100 IU/ml penicillin, 100 μg streptomycin, 0.1 mM β mercaptoethanol.

PBS : sterile solution of PBS (Ca⁺⁺ and Mg⁺⁺ free) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin culture medium Dulbecco's modified Eagle's medium supplemented with 15% PCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 0.1 mM β mercaptoethanol gelatin 0.1% gelatin in water

Trypsin : 0.25% trypsin, 0.04% EDTA in PBS (without Pen/Strep)

Freezing medium : 10% DMSO, 90% culture medium or FCS

Sex determination The sex of the isolated fibroblasts was determined by PCR (Pomp et al., /.

Anim. Sci. 1995; 73: 1408-1415). Two genes were amplified in a single reaction:

Sry (sex-determining region Y); the presence or absence of this gene determines sex;

Zfy (male) or Zfx (female); the amplification of these genes acts as a positive control for PCR.

The cells were found to be isolated from male foetus. Genetic modification

Muscle tissue derived fibroblasts were used to introduce gene markers: β-galactose gene and/or neomycin gene. The cell line, BEF-M, was transfected by electroporation with the following vectors: a)^~ 6PLacZ TINβS-MO linearized at a unique Xho I restriction site

(β-galactosidase-neomycin resistant fusion gene), or b) IRES-Neo linearized at a unique Mlu I restriction site (neomycin resistance gene).

2 x 10⁶ BEF-M cells were suspended in 0.4 ml of HeBs (20 mM

HEPES-NaOH pH 7.05, 137 mM NaCl, 5 mM KC1, 0.7 mM Na₂HP0₄, 6 mM glucose) containing 10 μg of linearized vector. The cell suspension was placed in a 4 mm gap electroporation cuvette and kept at room temperature for 10 min. A 200 V, 950 μF charge was then applied with a Gene Pulser II apparatus (Bio-Rad Laboratories). After 10 min incubation at room temperature the cell suspension was transferred to 10 ml of culture medium and plated onto 10 cm Petri dishes. The cells were re-fed culture medium containing 400 μg/ml Geneticin (Life Technologies) and selected for 8 to 14 days.

Following electroporation and selection with Geneticin, 18 6PLacZ TINβS-MO and 21 IRES-Neo stable transfectants were isolated. The positive clones were expanded and stored frozen in liquid nitrogen.

The positive 6PLacZ TLNβS-MO clones were analyzed for the expression pattern of β-galactosidase gene by staining with X-gal. One clone, where >90% of cells strongly expressed the marker gene, was chosen for nuclear transfer experiments.

EXAMPLE 4 Isolation of porcine adult fibroblasts Isolation and propagation

Ear samples were collected from adult male and female miniature swine. The tissue samples were briefly sterilized in 70% ethanol and washed in 3 changes of cold PBS containing 100 IU/ml penicillin, 100 μg streptomycin (PBS-P/S). The fibroblast cultures were established from skin as follows: The skin was disaggregated mechanically by chopping with scalpel blades followed by enzymatic digestion. For enzymatic digestion tissues were incubated for 5 hr (or up to 12 hr) at 4°C in 0.25% trypsin / 0.04% EDTA in PBS. After removing the excess of trypsin, tissues were incubated at 37°C for 30 min, culture medium was added and cells disaggregated by vigorous pipetting. Large, undigested pieces of tissue were allowed to settle by sedimentation. Supernatant containing single cells was decanted and the cells plated onto gelatinized (0.1% gelatin in water) Petrie dishes and cultured at 37°C in an atmosphere of 5% C0₂ in air. This was passage 0 (PO). The medium was changed every 24 hours and the cells cultured until confluent (between 5 to 8 days). The cells were then expanded by trypsinization and passaging 10⁵ to 10⁶ cells onto a 10 cm gelatinized Petrie dish in culture medium. Aliquots of cells at passage 0, 1, 2 were stored frozen in liquid nitrogen (in 10% DMSO, 90% culture medium). The culture medium was DMEM supplemented with 15% FCS, 2 mM glutamine, 100 IU/ml penicillin, 100 μg streptomycin, 0.1 mM β mercaptoethanol.

PBS : sterile solution of PBS (Ca⁺⁺ and Mg^ free) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin culture medium Dulbecco's modified Eagle's medium supplemented with 15% PCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 0.1 mM β mercaptoethanol gelatin 0.1% gelatin in water Trypsin 0.25% trypsin, 0.04% EDTA in PBS (without Pen/Strep) Freezing medium 10% DMSO, 90% culture medium or FCS

EXAMPLE 5 Gene constructs and transgenesis

The genetic modification of animal genomes can be broadly divided into random and targeted transgene integration. While random transgene integration suffers considerable limitations attributable to unpredictable site of integration effects, targeted transgene integration offers significant improvement in current loss of function applications and significantly greater opportunity in enabling planned modification of specific endogenous genes. To date, the generation of transgenic animals with targeted transgene integrations has been restricted to mice. Targeted mutations in mice have been possible due to the availability of ES cell culture systems which are not available for other species. ES cells, which are isolated from the inner cell mass of preimplantation mouse embryos, can be extensively propagated in vitro without losing their capacity to contribute to all cell types of an animal. The in vitro propagation of ES cells provides an extremely valuable opportunity to introduce transgenes and select clonal cell lines having the desired transgene integration event. The selected cell line can then be used as a genetic resource to establish an equivalent transgenic animal. An alternate route to ES cell-mediated transgenesis is to couple in vitro cell culture and genetic manipulation with nuclear transfer. Nuclear transfer differs from ES cell-mediated transgenesis in that a variety of cell types that can be cultured and genetically manipulated in vitro can be utilized as a genetic resource for the establishment of a new animal. For example, embryonic fibroblasts which can be propagated and genetically manipulated in vitro are not pluripotential and cannot contribute to the germline of an animal. However, the nuclei of genetically modified embryonic fibroblasts can be utilized as a genetic resource to establish a transgenic animal by reprogramming the developmental capability of that nucleus through nuclear transfer. Conversely, fertilized oocytes or single cells of in vitro or in vivo produced embryos can be cultured, genetically modified and used as a genetic resource for generating transgenic animals by nuclear transfer or aggregation with host embryos.

The introduction of targeted genetic modifications in specific genetic loci is facilitated by a process known as homologous recombination. Specific genetic changes can be introduced into a locus by flanking the new sequence with genomic sequences homologous to the target site. The homologous sequences recombine with the target endogenous gene and introduce the engineered mutation. To introduce specific targeted mutations in the germline of inbred MHC (SLA) miniature swine, we have engineered MHC (SLA) miniature swine genomic libraries and cloned and engineered a range of gene targeting vectors as follows. Figure 2 summarizes the steps taken for construction of the pGallaway targeting vector.

Endogenous genes are not limited to the examples shown. Gene targeting and nuclear transfer in swine and other animals would also be useful in cases of:-

1) Removal (knockout) of genes responsible for boar taint, 2) Disruption of the myostatin gene to increase muscle growth,

3) Growth modification: directing modified growth hormone construct into the growth hormone locus,

4) Using gene expression on the X and Y chromosomes to alter the ratio of males to females, 5) Addition of transgenes to increase disease resistance, interferon gene,

6) Production of human pharmaceuticals,

7) Disease free transfer of genetic blood lines,

8) Derivation of disease free swine from any genetic blood line,

9) Derivation of specific pathogen free (SPF) swine genetic blood lines.

MHC (SLA) Miniature swine genomic DNA library construction

Genomic DNA from a d/d miniswine shown to be homozygous at the αl,3-galactosyltransf erase locus by RFLP analysis was used to construct a genomic library. The same swine was used for derivation of the target cells used in these studies, assuring isogenicity and target locus. The lambda genomic library was made from DNA isolated from liver tissue using the Promega LambdaGEM-12 Xho I Half Site Arms (Promega Corp., Madison, WI) protocol with modifications. The genomic DNA was partially digested with Sau 3A I followed by partially filling-in of the Sau 3A I as described by Promega. The DNA fragments were then size fractionated by agarose gel electrophoresis using standard methodologies. The DNA was ligated to LambdaGEM-12 vector, which had already been digested with Xho I and dephosphorylated; and the first two nucleotides of the Xho I site filled in. The ligated DNA was packaged using the Stratagene Gigapack Gold Packaging Extract and mixed with E. coli host strain, KW251. The genomic library contained approximately 2 x 10⁶ independent clones.

Construction of vector, pBERT The unamplified library was screened with a 240 bp PCR fragment corresponding to nucleotides 607 through 847 of porcine α 1,3-galactosyltransferase gene (GT, Strahan et al., 1995, lmmunogenetics, vol. 41: 101-105, cDNA Sequence and Chromosome Localization of Swine αl,3~galactosyltransf erase, Genbank Accession Number L36152) using standard methodologies. Six bona fide porcine αl,3-galactosyltransf erase clones were identified and mapped by Southern analyses using standard methodologies. Two clones (#12 and 4) were selected for use in construction of the targeting vector, pGallaway.

The two lambda clones were subcloned as follows: lambda clone 12 was digested with Eco RI and the 10 kb fragment containing GT sequences upstream of exon 9 was isolated. This fragment was cloned into pUC19 Eco RI cut vector to generate pTCL12-45 (orientation 1) and pTCL12-40 (orientation 2). Lambda clone 4 was digested with Sac I and the 11.8 kb fragment containing GT sequences downstream of exon 9 was isolated, then cloned into pNEB 193 (NEB) Sac I cut vector to generate pTCR4-9. A 2.8 kb Eco Rl/Sac I fragment was also isolated from lambda clone 12 and cloned into pNEB 193 Eco Rl/Sac I cut vector to generate 4-2. This fragment contains 3' intron 8 sequences and 5' exon 9 sequences. Together, the 10 kb Eco RI fragment (clone 12), the 2.8 kb Eco Rl/Sac I (clone 12) and the 11.8 kb Sac I fragment (clone 4) form a 24.6 kb contiguous fragment containing the porcine GT extending from intron 7 through exon 9 into sequences 3' of GT. In order to introduce stop codons into the 5' end of exon 9, pBERT (Figure

3) was made. The plasmid, pTCL12-45, was digested with Eco RI and Sac I, the 7.5 kb fragment containing the GT region was isolated, a linker (comprised of oligonucleotides 5'-TCG ACT GTT TAA ACG GCC TCT ACG GCC TAG CT-3' (SEQ ID NO:l) and 5'-AGG CCG TAG AGG CCG TTT AAA CAG-3' (SEQ ID NO:2)) as shown in Figure 4 with cohesive Sal I and Sac I ends, which contained a unique Pme I and Sfi I was ligated to the fragment, followed by ligation to pUC19 Eco Rl/Sal I digested vector to generate pTCL12A. This vector lacks about 2.5 kb of the 5' end of the original lambda 12 clone. Stop codons were introduced into the 5' end of exon 9 by modifying 4-2. The plasmid, 4-2, was digested with Bse RI to drop out a 94 bp internal GT Bse RI fragment. This fragment was replaced with a synthetic fragment which was modified such that maximum isogenicity was maintained. Five bp changes were introduced into this synthetic Bse RI fragment. Two bp modifications changed the 5' Bse RI (within intron 8) to a unique Sal I site, while 3 bp changes created three in-frame stops within the first 50 bp of exon 9 (See Figure 5). The synthetic Bse RI fragment had cohesive Bse RI ends and was ligated to 4-2 Bse RI cut vector to generate 4-2A.

The vector, 4-2A, was digested with Eco RI and Sac I and the 2.8 kb Eco Rl/Sac I fragment containing the modified Bse RI region was isolated. Vector, pTCL12A, was digested with Eco RI, dephosphorylated, and ligated together with the 4-2A Eco Rl/Sac I fragment. The ligation reaction was first digested with Pme I, then with Sac I, and the 9-10 kb Pme I/Sac I fragment was purified. The vector, pTCR4-9, was digested with Not I, dephosphorylated, digested with Sac I and the resulting 11 kb Sac I/Not I fragment was purified. The vector, (SuperCos, Stratagene), was digested with Eco RI, which was filled-in with Klenow, then digested with Xba I and the 6.5 kb SuperCos ori fragment isolated. A second vector, SuperCos:stuffer, which contains the 11.4 kb Cla I fragment from EMBL3 cloned into the Cla I site of SuperCos, was digested with Xba I and Not I and the 12.6 kb Xba I/Not I fragment isolated.

The four purified fragments: 6.5 kb SuperCos ori (Eco RI)/Xba I; 12.6 kb SuperCos:stuffer Xba I/Not I; 9-10 kb TCL12A/4-2A Pme I/Sac I; and 11 kb TCR4-9 Sac I/Not I were ligated together in a two-step ligation. The TCL12A/42A fragment was first ligated to the TCR4-9 fragment and then the remaining two fragments were added to the ligation. The ligation mixture was packaged as described earlier. Clones were screened using standard methodologies and one correct clone, pBERT was used for further work.

Construction of pGallaway In order to clone to 5' region of GT, pTCL12-40 was digested with Eco RI, which was filled-in with Klenow, followed by digestion with Xho I. The resulting 6.5 kb fragment containing the 5' GT region was purified. The recipient vector (pOCUS/pgk-neo) is a modified pOCUS vector (Novagen) which contains a pgk-neo cassette cloned into the Hinc LI site of pOCUS. A partial Spe I digest was carried out on pOCUS /pgk-neo, the linearized Spe I vector purified, then digested with Xho I. The resulting 3.7 kb fragment containing the vector was purified and ligated to the 6.5 kb (Eco RI)/Xho I fragment from pTCL12-40 to generate pEddie.

The 3' GT region was cloned into pEddie as follows: pEddie was digested with Xho I and Not I, dephosphorylated and the 10 kb fragment purified. pBERT was digested with Xho I and Not I which removed the 18.1 kb GT region (with stop codon modifications) from the vector backbone. The unpurified 18.1 kb Eco RI/Not I fragment was ligated to the pOCUS/pgk-neo Eco RI/Not I vector. The ligated DNA was electroporated into competent £. coli TOP10 cells and individual colonies screened by standard methodologies. One correct pGallaway clone was identified and was used for further work. pGallaway can be used as both a replacement or insertion vector. Either the unique Xho I or Sal I sites within the GT region can be used to linearize pGallaway for use as an insertion vector. The entire 24.6 kb GT region can be removed from vector sequences by digestion with Sfi I and Not I. Several criteria were applied to the selection of an appropriate cell line in which to assess homologous recombination with the pGallaway vector. First, the line must be isogenic with the vector. Second, the line can be immortalized to permit sufficient passage between the initial transfection and subsequent analyses. Third, the line should express the α 1,3-galactosyltransferase gene, as expression will be required for the homologous recombination assay. Fourth, the line must be transfectable and selectable at reasonable frequencies. Fifth, to facilitate generation of an α 1,3-galactosyltransf erase null line, the line should be diploid. Aortic endothelial cells from the same miniature swine used to derive the genomic DNA library were immortalized with SV40 T antigen (Seebach et al., 1997, The 4th International Congress for Xenotransplantation, Nantes, France). One of the cell lines, PEDSV15, which satisfied the criteria listed above, was transfected by electroporation with the pGallaway targeting vector. The vector was linearized at a unique Xho I restriction site. 1 x 10 PEDSV15 cells were suspended in 0.8 ml of HeBS (20 mM HEPES-NaOH pH 7.05, 137 mM NaCl, 5 mM KC1, 0.7 mM Na₂HP0₄, 6 mM dextrose) containing 0.5 nM linearized pGallaway. The cell suspension was placed in a 4 mm gap electroporation cuvette and chilled on ice. A 270 V, 980 μF charge was then applied with a Gene Pulser apparatus (Bio-Rad Laboratories, Hercules, CA) and the cuvette returned to ice for 10 minutes. The cell suspension was then transferred to 10 ml of standard medium (M199 containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD) and pelleted. The cell pellet was resuspended and plated in enhanced medium (M199, 20% FBS, 100 μg/ml Endothelial Cell Growth Supplement (Collaborative BioMedical, Bedford, MA)) at approximately 6 x 10⁴ cell/cm² in various size plates to generate pools with varying numbers of stable transfectant clones, a limiting dilution series was performed to determine the stable transfection frequency for extrapolation of stable clones /plate. The cells were re-fed enhanced medium containing 400 μg/ml Geneticin (Life Technologies) and selected for 10-14 days.

RT-PCR Assay for Homologous Recombination

The genomic organization of the α 1,3-galactosyltransferase gene following a homologous recombination event between the chromosomal locus and the pGallaway vector is shown in Figure 6. The length of the α 1,3-galactosyltransferase homologous sequences in pGallaway (approximately 12 kb on either side of the changes introduced in exon 9) precluded use of a simple DNA PCR assay for identifying cells which had undergone a homologous recombination event. Additionally, to avoid the necessity of generating clonal lines of stable transfectants for assay, the assay had to specifically identify homologous recombination events within pools of stable transfectants also containing non-homologous insertions of the targeting vector.

Homologous recombination of the pGallaway targeting vector in endothelial cells should result in the generation of RNA transcripts containing the mutations (stop codons) introduced into the vector. These transcripts are most readily identified by RT-PCR using a forward primer from an α 1,3-galactosyltransf erase exon upstream of the vector sequences and a reverse primer specific for amplification of the expected mutant transcript. The forward primer chosen for these experiments was derived from wild-type exon 7 sequence. The reverse primer was derived from the mutated region of exon 9 in the pGallaway vector, and has a 3' terminal mismatch to the wild-type α 1,3-galactosyltransferase sequence. RNA was prepared from stable transfectant cells using the RNeasy Isolation Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. cDNA was prepared from RNA using the Superscript Preamplification System (Life Technologies), using random hexamers according to the manufacturer's instructions. cDNA was amplified in 100 PI reactions containing 10 mM Tris-HCl pH 8.3, 50 mM KC1, 10 μM each dNTP, 0.9 mM MgCl₂, and 25 μ/ml Amplitaq Gold polymerase (Perkin-Elmer, Norwalk, CT). Forward primer F238 5'-TTA CCA CGA AGA AGA AGA CGC-3" (SEQ ID NO:3) and reverse primer RD2 5'-TGC AGA TAT TCA GAA CTC CTC CT-3' (SEQ ID NO:4) were present at 200 nM. Amplification reactions were performed using a System 9600 thermocycler (Perkin-Elmer) with the following profile:

1) 95°C for 9 min.

2) 39 cycles of: 96°C for 2 sec

60°C for 30 sec 72°C for 30 sec 3) 72°C for 5 min.

Analysis of Targeting Efficiency

Two weeks following electroporation and selection with Geneticin aliquots of the stable transfectant pools were analyzed by RT-PCR as follows: To verify that amplifiable cDNA was prepared from the pools, the cDNA reaction was amplified with wild type primers F238 and R545 5'-AGA TGA CTT TGT GGC CAA CC-3' (SEQ ID NO:5), from porcine α 1,3-galactosyltransferase exons 7 and 9 respectively.

To ensure that the RNA pools were not contaminated with mutant template, the mock cDNA reactions were amplified with primers F238 and RD2. To identify pools containing cells producing the mutant transcript expected from the homologous recombination of pGallaway and the endogenous α 1,3-galactosyl transfer ase locus, the cDNA reactions were amplified with primers F238 and RD2. Initial analysis of 7 pools of approximately 500 stable transfectants each resulted in detection of the expected mutant template in all 7 pools. A subsequent analysis was performed on 6 pools of 30 stable transfectants (21 A-F) and 4 pools of 3 stable transfectants (22 A1,B1,C1,D1). The results of this analysis are shown in Figure 8. In panel A, ethidium bromide staining of PCR products generated from cDNA with the wild type F238 and R545 primers showed that amplifiable cDNA was produced from each pool.

In panel B, the pool cDNA preparations were amplified with primers F238 and RD2, for specific detection of product generated from the expected mutant transcript. Hybridization of Southern blots with an internal α 1,3-galactosyltransferase oligonucleotide probe revealed the presence of mutant transcripts in 2/6 pools of 30 (21 B,C). In panel C, mock cDNA amplified with primers F238 and RD2 confirmed the absence of contamination within any of the pools analyzed.

To eliminate any possibility that the PCR product generated from pools 21 B and 21 C was derived by low efficiency amplification of wild type RNA, the PCR product from these pools was digested with Ase I. Ase I should cleave the expected mutant product into fragments of 272 bp and 24 bp, but not cleave product derived from a wild type template (see Figure 7). The polyacrylamide gel analysis in Figure 9 clearly demonstrates that the PCR product from pools 21 B and C is cleaved, confirming the amplification of the mutant transcript expected from a homologous recombination event.

In summary, 2/6 pools of 30 stable transfectant clones were found to have mutant transcripts expected from a homologous recombination event, for a homologous recombination frequency of 1-2%.

To select for a cell line totally devoid of α 1,3-galactosyltransferase activity, i.e., a null cell line, we reasoned that screening the pools of clones for lack of binding to antibodies specific for the Galαl,3Gal epitope, synthesized by α 1,3-galactosyltransf erase might result in selection of such a null cell line. Accordingly, we subjected pools of the stable transformant cell lines to multiple rounds of selection using baboon xenogenic natural antibodies (XNAs) eluted from a Galαl,3Gal column matrix (Synsorb 90, Alberta Research Council, Canada) and rabbit complement. Following five rounds of selection, the vast majority of cells in one pool (2*3) were resistant to lysis. RT-PCR analysis was performed on RNA from the 2*3 pool prior to selection and after five rounds of selection. As expected from the size of the pools, only wild type PCR product was detected in the 2*3 pool prior to selection. Following selection, the vast majority of PCR product was that expected from the targeted allele. Small amounts of PCR product derived from the wild type RNA are compatible either with a small surviving sub-population of GT expressing cells or with a significant, but not total, loss of wild type RNA in the "null population". A FACS analysis was performed on the 2*3 line following 7 rounds of XNA/ complement selection, and the parental

PEDSV15 line to determine the relative levels of binding to Synsorb 90 or to the lectinlB4, which has binding specificity to Galαl,3Gal epitope. In both cases, the 2*3 cell line showed a greatly reduced level of binding, indicating that the cell line had a greatly reduced level of expression of the α 1, 3-galactosyltransferase activity.

Having demonstrated that gene targeting of the α 1,3-galactosyltransferase locus can be achieved in a porcine sortie endothelial cell line, it is understood that other cell types can be similarly modified, including but not limited to cultured cells, e.g., embryonic fibroblasts, or embryonic stem cells, or embryos.

The introduction of new genetic material and the subsequent selection of cells harboring the desired targeted integration requires expansion and clonal selection of each founder transgenic cell. A limitation to applying this processes in nuclear transplantation programs is the number of cell divisions which the transfected cell must undergo to provide sufficient material for molecular analysis of each transgenic colony and subsequent supply of nuclei for transfer. The great majority of cells suitable for in vitro genetic modification and subsequent nuclear transfer have limited in vitro propagation capacity. It is therefore desirable to utilize transfection and selection systems which generate and /or identify correctly targeted clones at high efficiency and with limited requirement for in vitro propagation.

A particularly efficient approach to selecting for correctly targeted clones is to use IRES gene trap targeting vectors. IRES gene trap targeting vectors provide a significant enhancement in gene targeting efficiency by eliminating a large proportion of random integration events. IRES gene trap targeting vectors rely upon functional integration into an actively transcribed gene (such as the target gene) for expression of the selectable marker. Random integrations into non-transcribed regions of the genome are not selected.

Widely expressed genes, such as the α 1,3-galactosyltransferase gene which is the principal target for gene deletion in xenotransplantation, is well suited to IRES gene trap targeting strategies in nuclear transfer programs. Figure 10 shows one α 1,3-galactosyltransf erase IRES gene trap targeting vector for functional disruption of α 1,3-galactosyltransferase gene expression. In a preferred embodiment, it may be desirable to remove the selectable marker cassette from the targeted locus to eliminate expression of the antibiotic resistance gene. One approach is to flank the IRES selectable marker cassette with suitable DNA sequences which act as recombination sites following the addition of a suitable site-specific recombinase. One example of a suitable recombinase site is the lox site which is specific for the Cre recombinase protein. Figure 11 shows one example of a recombinase sensitive α 1,3-galactosyltransferase IRES gene trap targeting vector for functional disruption of α 1,3-galactosyltransferase gene expression.

High efficiency gene targeting and selection has a significant advantage in that suitably stringent selection systems, such as the IRES gene trap targeting vectors, can eliminate the need for biochemical analysis of clonal cell lines. In this instance, individual nuclei from a pool of uncharacterized transgenic cells should generate offspring of the desired phenotype at a ratio equivalent to the selected pool. The elimination of clonal selection may be particularly useful where only limited in vitro propagation is desirable or possible. One such instance includes the culture of embryonic nuclei for nuclear transfer. Embryonic nuclei are more efficient than latter stage somatic cells for generating live born offspring by nuclear transfer, however, totipotential embryonic cells can not be cultured for extended periods for any other species than mice. Nuclear recycling of embryonic nuclei (see following) provides an opportunity to maintain, expand, and genetically manipulate multipotential cells from swine in vitro in the absence of typical ES culture systems.

Transgenes may be introduced into cultured cells or embryos by direct injection, electroporation, liposomes or calcium phosphate precipitation. Direct injection is the preferred method for individual embryonic cells while electroporation is more suitable for embryonic fibroblasts and embryonic stem cells. Typically, plasmid DNA for electroporation was linearized by restriction enzyme digest, ethanol precipitated and resuspended at 1.0 mg/ml in PBS (Oxoid, Unipath Ltd.). Following 2 hours culture in fresh medium, near confluent cells were dispersed by trypsinization, washed sequentially in culture medium and PBS (Oxoid, Unipath Ltd.), and resuspended at 1.4xl0⁸/ml in PBS (Oxoid, Unipath Ltd.) for immediate transfection. Routinely, 0.7 ml of cell suspension was mixed with 0.1 ml DNA containing solution and electroporated at 0.8 kV and 3.0 mFD using a Biorad Gene Pulser and 0.4 cm cuvettes. Transfections were plated on gelatinized tissue culture dishes at 5-8xl0⁴/cm² in growth medium for 16 hours prior to the addition of selection medium containing 200 mg/ml (active) G418 (Sigma). Single colonies were picked 8-10 days post transfection and transferred in duplicate into 24 well tissue culture plates for further expansion in growth medium containing 200 mg/ml G418 and subsequently in media without G418.

EXAMPLE 6 Elimination of α 1,3-galactosyltransferase activity in porcine foetal fibroblasts

Mesenchymal tissue derived fibroblasts were used to eliminate αl3-galactosyltransf erase activity. The cell line, PEM, was transfected by electroporation with the pGallaway targeting vector linearized at a unique Xho I restriction site. 1-4.8 x 10⁶ PEM cells were suspended in 0.4 ml of HeBs (20 mM HEPES- NaOH pH 7.05, 137 mM NaCl, 5 mM KC1, 0.7 mM Na₂HP0₄, 6 mM glucose) containing 0.5 mM linearized pGallaway. The cell suspension was placed in a 4 mm gap electroporation cuvette and kept at room temperature for 10 min. A 200V, 960 μF charge was then applied with a Gene Pulser II apparatus (Bio-Rad Laboratories). After 10 min. incubation at room temperature the cell suspension was transferred to 10 ml of culture medium and plated onto a number of 10 cm Petri dishes. The cells were re-fed culture medium containing 400 μg/ml Geneticin (Life Technologies) and selected for 8 to 14 days.

Following electroporation and selection with Geneticin aliquots of the stable transfectants were analyzed by RT-PCR as described in Example 3. Two sets of primers were used:

• wild type (primers F238 and R545) to verify that cDNA was transcribed successfully;

• mutant (primers F338 and RD2) to identify the cells producing the mutant transcript expected from the homologous recombination of pGallaway and the endogenous α 1,3-galactosyltransferase locus.

In two electroporation experiments, 7.6 x 10⁶ were transfected and 178 Geneticin resistant colonies isolated and analyzed for the elimination of one copy of the α 1,3-galactosyltransferase gene. Five individual targeted PEM clones have been identified, expanded and stored in liquid nitrogen. Four of these clones have been used for nuclear transfer so far.

The following strategy has been developed to identify and isolate primary clones harboring targeted integrations. To prevent extensive culture of targeted colonies, a "master" plate is stored frozen, while duplicate plate(s) are expanded and progressively smaller pools of colonies analyzed by RT-PCR for the targeted event.

The "master" plate is frozen as follows: When the cells reach confluency, the medium is removed and cells briefly washed with PBS, and 0.25% trypsin, 0.4% EDTA in PBS. The plate is then put on ice and 500 μl of cold freezing medium (10% DMSM, 90% FCS) added to each well. The plate is stored for up to 2 to 4 weeks at -75 °C or after 24 hr at -75°C transferred to liquid nitrogen. To thaw the cells, the plate is transferred to room temperature, 500 μl of warm culture medium added to each well and the cell suspension transferred to a new 24 well plate. The cells are cultured at 37°C in an atmosphere of 5% C0₂ in air.

EXAMPLE 7 Nuclear Transfer Activation Activation of the cytoplast was preferred for further embryonic development. Activation occurs during fertilization and is characterized by a series of Ca²⁺ oscillations, release of cortical granules, extrusion of a second polar body, pronuclear formation and ultimately cleavage. Development of an oocyte without fertilization by sperm is known as parthenogenic activation. Parthenogenic activation can be induced by various chemical and physical treatments such as ethanol, calcium ionophore and electrical pulses (Ozil, J.P., 1990). In the swine, electrical pulses have been shown to be more effective at induction of parthenogenetic development than ethanol (Saito et al., 1993). Electrical stimulation is therefore the preferred method of activation induction. Oocytes and reconstituted cytoplasts were activated by exposure to one or a series of electrical pulses (DC) at 90-120 V/mm for 30-90 μs (Prather et al., 1991).

The activated reconstituted oocytes were cultured in vitro overnight (24 hrs), or to the blastocyst stage (Day 6) of development (Example 6) prior to transfer into surrogate swine (Example 8). TABLE 3 Parthogenetic activation of porcine In Vitro and In Vivo matured oocytes using different methods

Fusion

The nuclear transfer procedure involved the reconstitution of an enucleated cytoplast with a karyoplast containing the donor nucleus. Cytoplasts were reconstituted by inducing membrane fusion with the karyoplast following treatment with chemical reagents, such as polyethyleneglycol (Czolowska et al., 1984), or by exposure to electrical pulses (Willadsen, 1986). Cytoplasts were also reconstituted by direct injection of the intact donor nucleus and karyoplast lysate into the cytoplast (Bromhall, 1975; Illmensee and Hoppe, 1981).

Electrofusion was the preferred technique for cytoplast reconstitution. The parameters required for successful fusion depends on the origin of the karyoplast and also whether the zonae pellucida was present on the cytoplast. When cytoplasts with an intact zonae pellucida were used, the karyoplast was inserted into the perivitelline space (PV) of the cytoplast between the oocyte membrane and zone pellucida, with all manipulation performed in cytochalasin B (Sigma Cell Culture, 5 μg/ml). To induce fusion, the cytoplast and karyoplast were placed between two platinum electrodes 0.5 - 1.0 mm apart in a solution of 0.3 M mannitol (Sigma Cell Culture), (Prather et al., 1989). For effective fusion to occur, the cytoplast and karyoplast were orientated in a perpendicular fashion so that the fusion plane of the two cells was parallel to the electrodes. An alignment current of 5-10 V AC (500-800 kHz for less than 10 s) was used to assist in orientation. When aligned, fusion was induced by a brief exposure to DC pulse (30-120 V/mm for 50 - 300 μs). Fusion of the cytoplast and karyoplast was observed within 15 -30 minutes by loss of membrane distinction between cells.

Cytoplast volume has been demonstrated to effect development of nuclear transfer embryos (Peura et al., 1997). It may therefore be beneficial to increase the cytoplast volume at fusion, especially when cytoplasts have been prepared by oocyte bisection (see Example 1). To increase cytoplast volume, zona pellucida free cytoplasts may be fused together before, after, or at the same time as karyoplast fusion using fusion parameters and conditions described above. Alternatively, optimal fusion parameters can be determined by the establishment and evaluation of isofusion contours. A comparison of our nuclear transfer procedures in the pig is shown in

Table 4. Many attempts at using existing published nuclear transfer approaches developed in sheep, (Campbell et al., 1997) and cattle (Cibelli et al., 1998) were unsuccessful for the pig.

The techniques that resulted in normal embryo development in a reconstituted "nuclear transfer" porcine embryo include

1) porcine oocytes are activated prior to enucleation, followed immediately by insertion of the donor nuclei and fusion 3 - 6 hrs after activation, and

2) modification of technique 1) where oocyte are enucleated (without activation), followed immediately by insertion of the donor nuclei with combined fusion activation occurring 3 - 6 hours after enucleation. TABLE 4 Efficiency of Nuclear Transfer in Pigs

Table 5 shows a summary of embryo transfer results for surgical transfer of reconstituted nuclear transfer porcine embryos produced by techniques of the present invention to synchronized recipients.

TABLE 5 Transfer of porcine NT embryos

CO c CD CO

H m en x m 10

c r m

M σ>

15

CO c

DO CO

O

X m

m

t

3 10 "KC" : modified nuclei donor

Only reconstituted NT porcine embryos have been transferred to recipients.

While a published nuclear transfer approach has produced live offspring in cattle (Cibelli et al., 1998) our invention provides a new way of producing nuclear transfer bovine embryos (see Table 6).

TABLE 6 In Vitro Cattle Nuclear Transfer

Karyoplast source

The technique developed for nuclear transfer has broad application to using donor nuclei other than foetal and embryonic cell lines. Reconstituted nuclear transfer porcine embryos were produced from transfer of adult pig fibroblast nuclei (see Table 7 and Example 2).

TABLE 7 Somatic Cell (Adult Cell) Nuclear Transfer in the Pig

Universal donor

The nuclear transfer technique was also assessed as to its suitability of the in vitro matured oocyte to service a universal donor.

Human fetal fibroblast were used as donor nuclei and fused to activated enucleated porcine oocytes. This demonstrated the utility of the nuclear transfer technique to provide development of nuclei to different species (see Table 8).

Table 8 Karyoplast Source (Human) for Porcine Nuclear Transfer

Serial nuclear transfer Alternatively, the activated reconstituted oocytes may be allowed to undergo one round of karyokinesis. The resulting nuclei can then be used for an additional round of nuclear transfer. This second round of nuclear transfer, or serial nuclear transfer, is postulated to improve the developmental capacity of the donor nuclei. Although the mechanism is unknown, re-exposure of the donor nuclei to molecular components in the cytoplasm of the cytoplast may assist in chromatin remodelling that is essential for nuclear reprogramming (Di Berardino, 1987; Kwon and Kono, 1996). To increase the number of nuclei available for serial nuclear transfer from one to two, the activated reconstituted cytoplasts were cultured in media with cytochalasin B (Sigma Cell Culture, 5 bagel) at 39°C in 5% C0₂ for 6 hours (Kwon and Kono, 1996). Following activation the donor nucleus re-entered the cell cycle and completed karyokinesis. The addition of cytochalasin B (Sigma Cell Culture) to the media inhibited the extrusion of the second PB resulting in the formation of two nuclei. Karyoplasts were prepared from both nuclei and consequently used for a second round of nuclear transfer by placing the karyoplast into the PV space of the cytoplast. The cytoplast in the second round of nuclear transfer was usually a zonae pellucida intact, enucleated PN stage fertilized oocyte (see Example 1). Fusion was induced in a similar manner to that used for the first round of nuclear transfer. Activation was not required. Embryo multiplication

To increase the number of embryos available for transfer following nuclear transfer, and therefore increase pregnancy rate, nuclear transfer may be performed using karyoplasts from 4-cell nuclear transfer embryos. In this example, the nuclear transfer procedure was used as a means of embryo multiplication. As cytoplasts reconstituted with karyoplasts at the same stage of the cell cycle demonstrate a higher developmental capacity (Cheong et al., 1993; Kwon and Kono, 1996), in this example, metaphase arrested karyoplasts were fused to cytoplasts prepared from enucleated metaphase (Mil) oocytes (see Example 1) using electiofusion and activation parameters described above. It was also possible to prepare karyoplasts from nuclei in the late G2 stage of the cell cycle (see Example 2). Nuclei within these karyoplasts were allowed to progress-through the cell cycle to mitosis or M phase, prior to fusion but were prevented from further progression through the cell cycle by incubation in nocodazole (Sigma Cell Culture, 1 μg/ml).

EXAMPLE 8 Isolation of porcine somatic cell lines for nuclear transfer Isolation

Nine foetuses were collected on day 28 of pregnancy from slaughtered DD miniature pig #12510 (G 5). Cell lines from four tissue types have successfully been isolated from each foetus:

• pig embryonic lung (PEL) fibroblasts

• pig embryonic mesenchymal (PEM) fibroblasts

• pig embryonic heart (PEH) fibroblasts • pig embryonic kidney (PEK) fibroblasts.

Propagation

Examples of all four cell types have been cultured for 9 passages (25 days). The results indicate that the growth curve (cumulative increase in cell number) and proliferative potential (growth ratio) of these cell lines is similar to that observed in previously isolated cell lines from commercial pig foetus.

Sex Determination

The sex of the isolated fibroblast cell lines was determined by PCR (Pomp et al., 1995).

Transfection efficiency

Electroporation is the method of choice for introduction of exogenous DNA into a fibroblast cell lines. However, other methods (e.g., lipofection) may also be used.

Electroporation parameters for each cell type have to date been optimized using control vector supplied by Peter Mountford. The results of these experiments were validated using the BTI construct (pGallaway). Other targeting constructs will be evaluated as they become available.

Elimination of α 1,3-galactosyltransferase activity in porcine embryonic fibroblasts Porcine embryonic mesenchymal tissue derived fibroblasts were used to confirm the feasibility of elimination of α 1,3-galactosyltransf erase activity. The cell line, PEM, was transferred by electroporation with the pGallaway targeting vector linearized at a unique Xho I restriction site as described in Example 3.1 x 10⁶ PEM cells were suspended in 0.4 ml of HeBS (20 mM HEPES- NaOH pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂HP0₄, 6 mM glucose) containing 0.5 mM linearized pGallaway.

The cell suspension was placed in a 4 mm gap electroporation cuvette and kept at room temperature for 10 min. A 270V, 960 μF charge was then applied with a Gene Pulser apparatus (Bio-Rad Laboratories). After 10 min incubation at room temperature the cell suspension was transferred to 10 ml of culture medium and plated onto 10 cm Petri dish. The cells were re-fed culture medium containing 400 μg/ml Geneticin (Life Technologies) and selected for 8 - 14 days. Following electroporation and selection with Geneticin aliquots of the stable transfectants were analyzed by RT-PCR as described in Example 3. Two sets of primers were used:

1) wild type (primers F238 and R545) to verify that cDNA was transcribed successfully;

2) mutant (primers F238 and RD2) to identify the cells producing the mutant transcript expected from the homologous recombination of pGallaway and the endogenous α 1,3-galactosyltransf erase locus.

Initial analysis of a pool of 11 Geneticin resistant colonies resulted in detection of the expectant mutant template. The results of this analysis is shown in Figure 13A. Ethidium bromide staining of PCR products generated from cDNA with the wild type primers F238 and R545, and with the mutant primers F238 and RD2 are shown in panel A. Both PCR fragments have the same size. However, cleavage treatment with Ase I yields two fragments of 272 bp and 24 bp in the mutant PCR product but no cleavage in the wild type PCR product (Figure 13B). The above results show that at least 1 out of 11 Geneticin resistant clones have mutant transcript expected from homologous recombination event. All 11 individual colonies have been stored in liquid N₂-

EXAMPLE 9 Culture of Embryos

Reconstituted oocytes were cultured in 20 μl drops of medium overlaid with paraffin oil (BDH Laboratory Supplies, UK or Sigma Chemical Company, Sigma-Aldrich Pty. Ltd.) under 5% C0₂ and reduced O_z (5-7%) atmosphere at 39°C. GI medium was used for the first 48 hr followed by G2 medium. The combination of GI and G2 medium and the reduced 0₂ greatly improved the quality of the early embryo and increased the number of cells that differentiated into inner cell mass cells.

Culture Media Porcine embryos were incubated in a number of different types of culture media. Two short term culture media were used for embryo collection, manipulation and embryo transfer:

1) Hepes (Sigma Cell Culture or Gibco BRL) buffered GI medium with 4 mg/ml BSA (Miles Pentex or Gibco BRL)

2) Hepes (Sigma Cell Culture or Gibco BRL) buffered MEM with 5 mg/ml BSA (Miles Pentex or Gibco BRL). The following long term culture media were used:

1) Whittens medium with 15 mg/ml BSA (Miles Pentex or Gibco BRL) 2) G1/G2 medium with 4 mg/ml BSA (Miles Pentex or Gibco BRL).

The embryos were cultured in micro drops of medium overlaid with mineral oil (BDH Laboratory Supplies, UK or Sigma Chemical Company, Sigma-Aldrich Pty. Ltd.), in 5%C0₂: 5%0₂: 90%N₂ atmosphere, at 38.5°C. The embryos were cultured for up to 7 days before embryo transfer to suitable recipient female.

The chemical composition of GI and G2 media is shown below. H-Gl is a hepes added version of GI medium. All chemicals are Sigma, except for BSA (Gibco or Miles Pentax).

GI from concentrated stocks Each reagent was added to MQ H₂0 as listed below, filtered, and stored at 4°C.

Stock 10 ml 50 ml

^■"^■GI 1.0 ml 5.0 ml

B 1.0 ml 5.0 ml ci 0.1 ml 0.5 ml

D 0.1 ml 0.5 ml

G 0.1 ml 0.5 ml

T 0.1 ml 0.5 ml

ED 01 ml 0.5 ml

Non-Essential AA (xlOO) 0.1 ml 0.5 ml

H₂0 7.4 ml 37.0 ml

BSA 30 mg 150 mg

H-Gl from concentrated stocks

Each reagent was added to MQ H₂0 as listed below, filtered, and stored at 4°C.

Stock 10 ml 20 ml 50 ml

B 0.16 ml 0.32 ml 0.8 ml

D 0.1 ml 0.2 ml 0.5 ml

G 0.1 ml 0.2 ml 0.5 ml

T 0.1 ml 0.2 ml 0.5 ml

ED 0.1 ml 0.2 ml 0.5 ml

Non-Essential AA (XlOO) 0.1 ml 0.2 ml 0.5 ml

HEPES 0.84 ml 1.68 ml 4.2 ml

H₂0 7.4 ml 14.8 ml 37.0 ml

BSA 30 mg 60 mg 150 mg

GI & H-Gl PREPARATION: Preparation of Concentrated Stocks:

Concentrated stocks were prepared by dissolving components in sterile MQ H₂0. Each stock was filtered and stored at 4°C for 3 months (Stock A^), 1 month (Stock D), or one week (Stocks B, D_G2, and G). Stock A Gl Component mM g/lOOml

(xlO concentration) NaCl 85.16 5.265

KCl 5.5 0.410

NaH₂P0₄2H₂0 0 5 0.078

MgS0₄ 7H₂0 1.0 0.246

Glucose 0.5 0.091

Penicillin - 0.06

Streptomycin - 0.05

NaLactate (50% syrup) 12.40 2.316

Stock B Component mM g/lOOml

(x 10 concentration! NaHC0₃ 25.0 2.101 Phenol Red 0.01^a

Stock C_G1 Component mM g/lOml

(x 100 concentration) NaPyruvate 0.32 0.0352

Stock D Component mM g/lOml

(x 100 concentration) CaCl₂2H₂0 1.8 0.265

Stock G Component mM g/lOml

(x 100 concentration) Glutamine 1.0 0.146

Stock T Component mM g/lOml

(x 100 concentration) Taurine 0.1 0.0125

Stock ED Component mM g/lOml

(x 100 concentration) EDTA 0.1 0.029^b

Stock HEPES Component mM g/lOml

(x 100 concentration) HEPES 20 5.958^c

Phenol Red 0.01^{a a} Usually only 0.0019 added ^b EDTA must be dissolved in 220 μl IM NaOH prior to adding H₂0 ^c Adjust to pH 7.4 with NaOH dissolved in media. Use pH strips as an initial indicator, then confirm with pH meter. G2 PREPARATION:

Preparation of Concentrated Stocks:

Concentrated stocks were prepared by dissolving components in sterile MQ H₂0. Each stock was Altered and stored at 4°C for 3 months (Stock A^), 1 month (Stock D), or one week (Stocks B, D_G2, and G).

Stock A_G1 Component mM g/lOOml

(xlO concentration) NaCl 85 16 5.265

KCl 5.5 0.410

NaH₂P0₄2H₂0 0.5 0.078

MgS0₄7H₂0 1.0 0.246

Glucose 3.15 0.568

Penicillin - 0.06

Streptomycin - 0.05

NaLactate (50% syrup) 11.74 2.193

Stock B Component mM g/1001

(x 10 concentration) NaHC0₃ 25.0 2.101

Phenol Red o or

Stock C_G2 Component mM g/lOml

(x 100 concentration) NaPyruvate 0.10 0.011

Stock D Component mM g/lOml

(x 100 concentration) CaCl₂2H₂0 1.8 0.265

Stock G Component mM g/lOml

(x 100 concentration) Glutamine 1.0 0.146 Usually only O.OOlg added

G2 from concentrated stocks

Each reagent was added to MQH₂0 as listed below. The pH was measured and adjusted to pH 7.4 with NaOH (approx 50 μl IM NaOH/ 10ml). The medium was filtered and stored at 4°C. Osmolarity should be 269 mOsm. Stock 10 ml 50 ml

B 1.0 ml 5.0 ml

CG2 0.1 ml 0.5 ml

D 0.1 ml 0.5 ml

G 0.1 ml 0.5 ml

Non-Essential AA (xlOO) 0.1 ml 0.5 ml

Essential AA (x50) 0.2 ml 0.5 ml

H₂0 7.4 ml 37.0 ml

BBSSAA 3300 mmgg 150 mg

EXAMPLE 10 Generation of Transgenic Offspring by Nuclear Transfer

Viable offspring can be generated from transfer of embryonic or somatic cell nuclei to enucleated oocytes. Modification of this technology to include transgenic donor nuclei allows the generation of transgenic animals.

To demonstrate the generation of transgenic animals from nuclear transfer we have developed a novel approach using a mouse model. This approach involves the use of a unique transgenic mouse that had a nuclear localized lacZ-neoR expression as a result of random gene-trap integration of pG TRES-βgeopA into what appears to be an ubiquitously expressed gene. As a result all cells from this mouse, including embryonic blastomeres, demonstrate distinctive blue nuclei when treated with the X-gal substrate. In this example, karyoplasts were prepared from embryonic blastomeres of this mouse and used in nuclear transfer. Blastocysts that developed following in vitro culture of the reconstituted oocyte, were either treated with X-gal or transferred to a recipient female. All nuclei in the X-gal treated blastocysts displayed distinctively blue staining (Figure 12.1). Tissue samples from pups born following blastocyst transfer also displayed distinctively blue staining (Figure 12.2 shows nuclear transfer pups with litter mates and Figure 12.3 shows X-gal stained tissues (tail tips) from nuclear transfer pups and litter mates). As this staining pattern is characteristic of the transgenic mouse from which the donor nuclei were isolated, this novel approach clearly confirmed that transgenic offspring can be generated from transgenic karyoplasts (nuclei) without loss of transgene expression.

EXAMPLE 11 MHC (SLA) Miniature Swine

A unique pig strain was used in the nuclear transfer program. The National Institute of Health (MHC /SLA) miniature swine is an inbred stiain of swine with a genetically defined major histocompatibility complex. A breeding program was commenced in 1972 to develop a strain of inbred (MHC/SLA) miniature swine to increase homozygosity at loci associated with the swine lymphocyte antigen complex (Sachs et al., 1976). Specific antibodies for each of the MHC (SLA) miniature pig strains have been isolated. Use of these strain specific antibodies allows unequivocal detection of contribution of karyoplast nuclei to the development of any pig produced by nuclear transfer.

EXAMPLE 12 Embryo Transfer in Outbred and MHC (SLA) Miniature swine.

Oestrous Synchronization

Several methods were developed to achieve synchronization of both outbred and MHC (SLA) miniature swine. Selection of one particular method depends on the age and size of the animal, and on available facilities.

Method 1: Corpus luteum regression following Prostaglandin treatment

Recipient swine, 25 - 40 days pregnant (as confirmed by ultrasound or non return to estrus) were selected for embryo transfer. The following procedure was used to induce synchronization:

Day 1 (am) intramuscular injection with 4 ml of Juramate (Cloprostenol, Pitman-Moore Australia Ltd.) or Prostaglandin F2α analogue.

Day 2 (am) intramuscular injection with 2 ml of Juramate (Cloprostenol) or Prostaglandin F2α analogue followed by 500 RJ PMSG (Folligon, Intervet (Aust) Pty. Ltd. or Pregnecol, Horizon Technology Pty. Ltd.). Day 5 (am) Intramuscular injection with 500 IU of hCG (Chorulon, Intervet (Aust) Pty. Ltd.).

Day 6 Detection of estrus (standing heat). Day 7 One cell embryo transfer. Day 11 Blastocyst transfer.

Method 2: Natural cycling

Unstimulated, natural cycling recipient pigs were selected for embryo transfer. One cell embryos were transferred 24 hours after detection of estrus. Blastocysts were transferred 5 days after detection of estrus.

Method 3: Extended Progesterone treatment.

Natural cycling pigs were treated with Altrenogest (Regumate, Roussel-Ulcaf, Paris, France) for 14-18 days (15-20 mg/animal/day) to synchronize estrus cycles. Twenty four hours after removal of Regumate (Altrenogest, Hoechst-Roussel), swine were injected with 500 IU of PMSG (Folligon, Intervet (Aust) Pty. Ltd. or Pregnecol, Horizon Technology Pty. Ltd.) followed two days later injection of 500 IU of hCG (Chorulon, Intervet (Aust) Pty. Ltd.).

Method 4: Synchronization of estrus of sows after lactation or weaning.

Recipients were synchronized for embryo transfer at weaning using the following procedure: Day 1 Weaning (removal of piglets).

Day 2 (am) intramuscular injection of 500 IU PMSG (Folligon, Intervet (Aust) Pty. Ltd. or Pregnecol, Horizon Technology Pty. Ltd.).

Day 5 (am) intramuscular injection of 500 IU hcG (Chorulon, Intervet (Aust)). Day 6 Detection of estrus (standing heat).

Day 7 One cell embryo transfer.

Day 11 Blastocyst transfer. Surgery Standard surgical procedures were used. Following induction of anaesthesia with Thiopentone, surgical anaesthesia was maintained with either halothane (fluothane, Zeneca, ICI Australia Operations Pty. Ltd.) or isofluorane (Forthane, Abbott Labs, UK) in oxygen. Reproductive tracts were exteriorized by midline laparotomy.

Oocytes or pronuclear stage embryos were collected through a modified teflon catheter inserted into the oviduct via the infundibullum. Embryos were collected by retrograde flushing with flushing medium (Dulbecco's phosphate buffered saline (Gibco BRL, Life Technologies Inc., NY or Trace Biosciences Pty. Ltd. [D-PBS] supplemented with 1% fetal calf serum (CSL Limited, Aus), calcium and magnesium).

Embryos were transferred in short term culture medium. One to four cell embryos were transferred to the oviduct of a recipient using a torn cat catheter and 1 ml syringe. Eight -cell to blastocyst stage embryos were transferred to the uterus of the recipient using the same equipment.

Recipients were synchronized to the development stage of the embryos transferred. Improved pregnancy rates were achieved by asynchronous transfer. The estrous cycles of recipient female pigs were programmed 24 hours behind embryo development. Approximately 67% of recipients farrow following such embryo transfer.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

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69, 19-34. 6) DiBerardino, M.A. (1987) Genomic potential of differentiated cells analysed by nuclear transplantation. Am. Zool. 27, 623-644. 7) Funahashi, H., Cantley, T., Stumpf, T.T., Terlouw, S.L., and Day, B.N.

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8) Funahashi, H., Cantley, T.C., Stumpf, T.T., Terlouw, S.L. and Day, B.N. (1994b) Use of Low-Salt Culture Medium for in Vitro Maturation of Porcine Oocytes is Associated with Elevated Oocyte Glutathione Levels and

Enhanced Male pronuclear Formation after in Vitro Fertilization. Biology of Reproduction 51:633-639.

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54:1412-1419.

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Claims

1. A method of generating an animal embryo, which method includes providing a donor nucleus, and a recipient cell; removing the nucleus from the recipient cell; introducing the donor nucleus into the enucleated cell to produce a couplet; and maintaining the couplet in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape.

2. A method according to Claim 1 further comprising an activation step.

3. A method according to Claim 2 wherein the couplet is subsequently subjected to a cell fusion step.

4. A method according to Claim 1 wherein the couplet is subsequently subjected to a cell fusion/activation step.

5. A method according to Claim 2, 64, or 65 wherein the animal embryo is a murine, bovine, ovine or porcine embryo.

6. A method according to Claim 5 wherein the embryo is a porcine embryo.

7. A method according to Claim 6 where the donor is selected from the group consisting of embryonic, foetal and adult cells.

8. A method according to Claim 7 wherein the donor nucleus is from a porcine animal.

9. A method according to Claim 8 wherein the donor nucleus is from an MHC (SLA)-miniature swine.

10. A method according to Claim 2, 64, or 65 wherein the recipient cytoplasm is prepared from in vivo or in vitro produced oocytes.

11. A method according to Claim 10 wherein the oocytes are oocytes which have been subjected to an in vitro maturation step.

12. A method according to Claim 11 wherein the in vitro maturation is conducted in the presence of estrus cow serum.

13. A method according to Claim 1 wherein the couplet is maintained in a suitable medium for a period of approximately 3 to 8 hours.

14. A method according to Claim 13 wherein the couplet cytoplasm is maintained in a suitable medium for a period of approximately 4.5 to 6 hours.

15. A method according to Claim 1 wherein the donor nucleus is introduced into the enucleated cell substantially immediately after enucleation.

16. A method according to Claim 15 wherein the nucleus is removed from the activated cell via an incision site and the donor nucleus is introduced into the enucleated cell through the same incision site.

17. A method according to Claim 16 wherein the donor nucleus is introduced under the zona pellucida.

18. A method according to Claim 1 wherein the donor nucleus is genetically modified by random transgene integration or targeted transgene integration in an endogenous gene in the donor nucleus.

19. A method according to Claim 18 wherein the endogenous gene in the donor nucleus is modified by introducing into said donor nucleus a DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations, such that there is homologous recombination between the construct and the endogenous gene.

20. A method according to Claim 19 wherein the animal nucleus is a porcine nucleus and the endogenous gene is the ╬▒ 1,3-galactosyltransferase gene.

21. A method according to Claim 20 wherein the DNA constiuct is a plasmid selected from the group consisting of pGallaway, pBERT I and pBERT II, or a functionally equivalent plasmid

22. A method according to Claim 1 wherein the couplet is cultured in vitro to produce the transgenic animal embryo and then the transgenic animal embryo is transferred to a surrogate for subsequent development into a transgenic animal.

23. An animal embryo when produced by a method according to Claim 1.

24. A method of generating a transgenic animal embryo which method includes providing a donor nucleus which has been genetically modified to eliminate an undesirable activity or to provide for a desirable activity, and a recipient cell; transferring the donor nucleus to the recipient cell to produce a couplet; and generating a transgenic animal embryo from said couplet.

25. A method according to Claim 24 wherein the transfer step includes removing the nucleus from the recipient cell; introducing the donor nucleus into the enucleated cell to produce a couplet; and maintaining the couplet in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape.

26. A method according to Claim 25 including the preliminary step of subjecting the recipient cell to an activation step; and subsequently removing the nucleus from the activated cell.

27. A method according to Claim 24 wherein the transgenic animal embryo is a porcine embryo.

28. A method according to Claim 27 wherein the donor is selected from the group consisting of embryonic, foetal and adult cells.

29. A method according to Claim 28 wherein the donor nucleus is from a MHC (SLA) miniature swine.

30. A method according to Claim 24 wherein the recipient cell is prepared by enucleation of in vivo or in vitro produced oocytes.

31. A method according to Claim 30 wherein the oocytes are oocytes which have been subjected to an in vitro maturation step.

32. A method according to Claim 31 wherein the in vitro maturation is conducted in the presence of estrus cow serum.

33. A method according to Claim 29 wherein the recipient cell is from a MHC (SLA) miniature swine.

34. A method according to Claim 26 wherein subsequent to the transfer step, the membrane fusion of the couplet is induced.

35. A method according to Claim 34 wherein the membrane fusion is induced by exposing the couplet to electrical pulses or polyethylene glycol.

36. A method according to Claim 34 wherein the recipient cell is activated by exposure to an agent selected from the group consisting of ethanol, calcium ionophore and electrical stimulation.

37. A method according to Claim 24, 25, or 26 wherein the donor nucleus is from an embryo that is itself a product of nuclear transplantation.

38. A method according to Claim 37 wherein the donor nucleus is from an embryo that is a product of serial nuclear transplantation.

39. A method according to Claim 24, 25, or 26 wherein the donor nucleus is genetically modified by random transgene integration or targeted transgene integration in an endogenous gene in the donor nucleus.

40. A method according to Claim 39 wherein the endogenous gene in the donor nucleus is modified by introducing into said donor nucleus a DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations, such that there is homologous recombination between the construct and the endogenous gene.

41. A method according to Claim 24 wherein the undesirable activity is xenoantigenicity.

42. A method according to Claim 41 wherein the animal nucleus is a porcine nucleus and the endogenous gene is the ╬▒ 1,3-galactosyltransferase gene.

43. A method according to Claim 42 wherein the DNA constiuct is a plasmid selected from the group consisting of pGallaway, pBERT I and pBERT II or a functionally equivalent plasmid.

44. A method according to Claim 34 or 67 wherein the couplet is cultured in vitro to produce the transgenic animal embryo and then the transgenic animal embryo is transferred to a surrogate for subsequent development into a transgenic animal.

45. A transgenic animal embryo when produced by a method according to Claim 24, 25, or 26.

46. A transgenic porcine embryo produced by a method according to Claim 24, 25, or 26.

47. A tiansgenic animal produced by a method according to Claim 38.

48. A transgenic animal according to Claim 40 wherein the expression of an endogenous gene has been deleted.

49. A transgenic animal descended from a transgenic animal according to Claim 47.

50. A transgenic animal descended from a transgenic animal according to Claim 48.

51. An organ derived from a tiansgenic animal according to Claim 47.

52. Tissue derived from a transgenic animal according to Claim 47.

53. Cells derived from a transgenic animal according to Claim 47.

54. A DNA construct for modifying an endogenous gene in an animal nucleus, said DNA construct including a nucleic acid sequence which is substantially isogenic to at least one or more portions of the endogenous gene and includes one or more mutations.

55. A DNA constiuct according to Claim 54 wherein the animal nucleus is a swine nucleus and the endogenous gene is ╬▒ 1,3-galactosyltransf erase.

56. A DNA construct according to Claim 55 which is pGallaway or a functionally equivalent plasmid.

57. A DNA construct according to Claim 54 further including a selectable marker such that functional integration of said DNA construct into said endogenous gene results in expression of the selectable marker.

58. A DNA construct according to Claim 57 wherein the animal nucleus is a swine nucleus and the endogenous gene is ╬▒ 1,3-galactosyltransferase.

59. A DNA construct according to Claim 58 which is pBERT I or a functionally equivalent plasmid.

60. A DNA construct according to Claim 59 further including recombination sites flanking said selectable marker such that addition of a recombinase results in removal of the selectable marker by recombination at the recombination sites.

61. A DNA constiuct according to Claim 60 wherein the animal nucleus is a swine nucleus and the endogenous gene is ╬▒ 1,3-galactosyltransferase.

62. A cell or cell line containing a DNA constiuct according to Claim 54.

63. A method according to Claim 25 wherein, subsequent to the transfer step, membrane fusion/activation of the couplet is induced.

64. A method of generating an animal embryo, which method includes providing a donor nucleus, and a recipient cell; removing the nucleus from the recipient cell; maintaining the enucleated cell in a suitable medium for a period sufficient to allow the cell to recover a substantially normal shape; and transferring the donor nucleus to the enucleated cell by direct injection.

65. A method according to Claim 64, further comprising an activation step.

66. A method according to Claim 64 or 65 wherein the enucleated cell is maintained in a suitable medium for a period of approximately 3 to 8 hours.

67. A method according to Claim 64 or 65 wherein the donor nucleus has been genetically modified to eliminate an undesirable activity or to provide for a desirable activity.

68. A tiansgenic animal produced by a method according to Claim 64, 65, or 67.

69. A transgenic animal descended from a tiansgenic animal according to Claim 68.

70. The method according to Claim 65, wherein the activation step is after the direct injection.

71. The method according to Claim 65, wherein the activation step is prior to removing the nucleus from the recipient cell.

72. The method according to Claim 2, wherein the activation step is after the introduction of the donor nucleus into the enucleated cell.

73. The method according to Claim 2, wherein the activation step is prior to removing the nucleus from the recipient cell.