WO1999021415A1 - 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 identioal offepring 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 paπicular, cloning techniques known In the prior art for ovine and bovine animals (see for example WO97/07669 and W095/17500) 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 nucleuβ from the recipient cell; introducing the donor nucleus into the enucleated cell to produce a A tt±.1- rβcθB-stitmsgreeli; and

^• maintaining the rβββnβttoted^ett- In a suitable medium for a period

Applicants have found, in this preferred embodiment, improved results where a preliminary activation step is undertaken. coo^ie.-^* Preferably the rαuuiuilϊϊulϊ j Lull is subsequently subjected to cell fusion.

Where the preferred preliminary activation step ia not utilised the recenstitαto^'d'tell may be subjootod to a cell fueion/activation step. For example, where electrical pulses are utilised for ceil 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 activily, and a recipient cell; transferring the donor nucleus to the recipient cell to produce a eoκ* .+- rcsaπsWutad call; and C _.ou -^β _Λlι _• generating a transgenic animal embryo from said reeen mύtitutac. eell.

The animal embryo may be of any type, and iπoludec bird, fiεh, reptile and mammalian (including ungulato 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 tho transfer step includes removing the nucleus from the recipient call; introducing the donor nucleus into the enucleated cell to produce a MttohwiiutB-. call; and maintaining the wyϋnia utϋϋ uall 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 plaεma 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 (1CM) cells, embryonic disc (ED) celiε or primordial germ cells (PGC)] may be used. A cell line derived from an embryonal tumor may be used (eg. embryonal carcinoma (EC) or yolk sac tumor cells). Adult celts such as fibroblasts may also be used. In this case the whole cell may be fused to the recipient cytopiasm. The recipient ceil may be of any suitable type and from any suitable species. Cytoplasts may be prepared from in vivo or in vrtro produced oocytes.

Cytoplastε may be prepared from oocytes arrested in the second metaphase of meiotic maturation (Mil oocytes). Other sources of cytoplasts include zygotes, fertilised oocytes. and 2-cell blastomeres.

Cytoplast preparation involves the removal of the nucleus in a process referred to as enucleatlon. 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 microfiiamβnt inhibitor, for example cytochalasin B (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.), that relaxes the cytoskeleton and allows the removal of a pomon of membrane enclosed cytoplasm containing the pronuclei or metaphase plate. Alternatively, nonphyslcal approaches such as inactivation of the

cytoplasts e ore, after or at approxmatey t e same me as onor nuc eue _A An animal embryo may be generated from the iLuuilUllt .J Lull by any εuilable method. Embryonic development may be initially in vitro and subsequently in α surrogate. Thus, the aMaa*w.u.^ge eβll may be initially cultured

5 in vitro to produce an embryo and then the embryo may be transferred to a

^■_JTU W- -^surrogate for subsequent development Into an animal. In vitro culture of the ct * aas a nntit od^* eiite may be in any suitable medium.

Activation occurs during fertilisation when the penetrating sperm triggers the resumption of meiosis. Activation is characterised by calcium oscillation,

10

Aotivation ietoeriormβd prior toϊransfer ot the donor nuclei.

In a preferred embodiment of this aspoot of the present invention, the donor 15 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, εerial nuclear transfer is postulated to improve the developmental capacity 20 of transplanted nuclei by allowing specific molecular components in the oocyte to assist ;in chromatln 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, nuolear reprogramming and embryonic development

25 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 ot 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 haε been the eubject of teohnioal 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 βn 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 α1 ,3-galactosyltransferase gene may be modified in transgenic swine. This gene is non-funotional in humans and so it is no longer expreeβed. 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 α1 ,3-galactosyltransferaεe 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 y' 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 G7-Θ8% 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 idcntioal. Accordingly, it is preferable to use targeting DNA derived from cells as closely related as possible to the ceil 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 isogonio or εubstantiaily ieogβnic 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 hybridise with each other under the most stringent hybridisation 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 endonucleasos). The β 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 βtill hybridise with each other under the most stringent conditions (βee Sambrook, J., et al., 1989) and that they will only rarely exhibit restriction fragment length polymorphism (RF P) 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 cf the present invention there is provided a DNA construct tor 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 α1 ,3-gaiactosyltransferase gene. In a particuiariy preferred embodiment of this aspect of the invention, the DNA construot 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 0.1 ,3-gatactosyltransferase gene and other mutations to that gene are contemplated.

The introduction of new genetic material and the subsequent selection of cells harbouring the desired targeted integration requires expansion and cional 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 oeli 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 utilise 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 incoφorated herein by reference. The

IRES :gβnβ trap targeting vector may be selected from IRES-neo, IRES-lacZ,

(TAA3) IRES-lacZ, (TAA₃) IRES-lacZ lox neo-tk lox. (TAQ₃) IRES-lacZ mclneo. SA lacZ-IRES neo, SA (TAAa) IRES-nuclear lacZ, SA (TAA3) IRES-nuciear lacZ lox Qprt lox, IRES-βgeo, (TAA₃) IRES-βgeo, SA IRES-βgeo SA Optimised

IRES-βgeo, IRES-nuclear βgeo, SA IRES-nuclear βgeo, SA (TAA₃) IRES-nuclear βgeo. SA Optimised IRES-nuclear βgeo, IRES-∑eo, SA IRES-zeo, IRES-hph, SA

IRES-hph, IRES-hph-tk, IRES-bsd, SA IRES-bsd, IRES-puro. IRES gene trap targeting vectors provide α 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 α1 ,3-galactosyltraπsferase gene. In a particularly preferred embodiment of this aspect of the invention, the DNA construct is pBERT 1 , as hereinafter described or a functionally equivalent piasmid.

In a preferred embodiment, it may be desirable to remove the selectable marker cassette from the targeted locus to eliminate expression of the eg. 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 Ore recombinase protein. Another example of a suitable recombinase is the FLP/FRT recombinase system (O'Gorman, S., et l., 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 nucleio aoid sαquenoe 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 recombinaee 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 ia a ewine or murine more preferably a swine α1 ,3-galactosyltransferase gene. In a particularly preferred embodiment of this aspect of the invention, the

DNA construct is pBERT 11 , 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 cioπal cell lines. In this instance, individual nuclei from a pool of uncharacterised transgenic ceils should generate offspring of the desired phenotype at a ratio equivalent to the selected pool. The elimination of olonal 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 bom offspring by nuclear transfer. However, totipotentiai 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 ceils from animals in vitro.

The DNA constructs according to this aspect of the invention may be engineered in bacteria and then introduced into the ceils. 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 identioal) not allogenic (genetically dissimilar) at the genetic locuε that is to be targeted.

In a further aspect of the present invention there ie provided an animal embryo or tranεgenic 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 ceil 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 εwine.

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 ie 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 1 A shows an overview of a nuclear transfer procedure according to the present invention in which activation occurs prior to enucieation which is then

followed by a quiescent period of, e.g. 6 hours and ther fusion.

Figure 1B 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 thenjcomblned fuslon/actlvation.

iFigure 2 summarises the steps taken for construction of the pGallaway targeting vector. < «.₍•**; /*<?C » o < -*>•«>-;<.«,-, 7j «_ C*J _* %_ G»/l«.«.Ji _{V t}

Figure 3 ie 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 organisation of the swine α1 ,3-galactoβyltransferase gene following a homologous recombination event between the chromosomal locus and the 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 1 digestion of mutant PCR products.

Figure 10 is a map of pBERT 1.

Figure 11 is a map of pBERT 11.

Figure 1 shows transgenic animals generated by nuciear 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 1 digestion.

Figure 14 shows a strategy developed for sorecning 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 HC (SLA) inbred miniature swine. Whilst applicant does not wish to bo restricted by theory, it is thought that the use of the MHC (SLA) inbred miniature swine oocytes as a source of cytopiasts 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 synchronise 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. Synchronisation of the estrous cycle was achieved by the following treatments: 1. administration of synthetic progesterone (eg. Regumate, Altreπogesl. Hoechst-Roussel); 2. prostaglandin or prostaglandin analogue injection of a pregnant swine; 3. removal of offspring at weaning. Natural cycling swino was also used. Superovuiation with follicle etimuiating hormone (FSH) waβ 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. Ovuiation time was controlled with the injection of human chorionic gonadotrophin (hCG (Chorulon, Intervet (Aust) Pty. Ltd., 500 IU). Unfertilized metaphase 11 oocytes were surgically collected 2-16 hours after ovuiation. In vitro oocyte production

Pigs

Ovaries were collected from slaughtered gilts and transported to the laboratory at 37^βC in Saiine (0.9% Sodium Chloride) (HSA, Baxter AHB1324) supplemented with 10μl ml peniciiiin-εtreptomycin solution (contains 5,000 iu/ml penicillin G sodium and 5,000μg ml streptomycin sulfate, Gibco BRL, Life

Technologies).

Oocyte cumulus comploxeε (OCC) were aspirated from follicles 2-6mm in diameter. OCC with uniform cytoplasm and compact cumulus cell mass were collected, nnsed in Dulbecco'ε phosphate buffered saline (Gibco BRL. Life

Technologies Inc.) supplemented with 0.3% bovine serum aibumin (ICN

Biomedicals Inc.) and cultured in one of the following media:

Medium 1 Medium 199 (Sigma Cell Culture, Slgma-Aldrich Pty. Ltd.)

Medium 2 Modified Whittenε medium (Whitten, W.K. and Bigger, J.D., 1968, Funahashi, H ot a/., 1994b, 1996)

Medium 3 Modified G1 medium

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

TABLE 1

In Vitro maturation of porcine oocytes

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, Slgma-Aldrlch Pty. Ltd.). at 38.5°C* under 5%CO_∑ 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 Follicl Stimulating Hormone ( from porcine pituitary, Sigma-Aldrich Pty. Ltd.) The benefits of adding of Eetrus cow serum is shown in Table 2.

TABLE 2

Effects of different aera on the in vitro maturation of porcine oocytes

^•ECS, Estrus Cow Serum; FBS, Fetal Bovine Serum " Basic medium, TCM 199; Maturation time -48-52 hrs

5 Cattle

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

Mlcrosurglcal Eπucleation or Bisection of nuclear material

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

Enuoieation by microsurgery

Prior to enucleation the oooytos were denuded of cumulus cells by gentle pipetting after 2 minutes vortexing in 0.1% hyaiuronidase (Sigma Cell Culture ,

15 Sigma-Aldrich Pty. Ltd., 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 Ceil Culture, Sigma-Aldrich Pty. Ltd. 5μg/ml) for approximately 10 minutes at 38.5°C followed by washing in Hepes (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd. or Gibco BRL, Life Technologies Inc.) buffered M199, G1 or other medium

20 (listed above). The enucleation was performed in a 20 μl drop of Hepes buffered medium (M1Θ9 or G1) containing the cytoεkelβtal inhibitor, cytochaisin B (Sigma 16

Cell Culture, Sigma-Aldrich Pty. Ltd. 1 ,0 - 7.5 μg/ml). Using the micromanipuiation 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 enucieation 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 oooytc, zona pellucida and ooiemma (cytoplasmic membrane) was important. Failure to have both pipettes and oocyte aligned resulted in lysis of the oocyte. Having penetrated the zona and ooiemma, 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 1/6) of the oocytes cytoplasm and the first polar body. Successful enuoieation 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 al 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 visualisation and orientation of the metaphase II plate. Following enucieation the oocyte and confirmation of enucleation was identical to the methods described above.

Cytoplast from the second round of nuclear transfer were prepared from in vivo sourced zygotes. Donor female gilts or βwine were synchronised and supcrovulated as described above. Females were mated with males twice, 24 hours and 32 hours after hCG (Choruion, Intervet (Aust) Pty. Ltd.) injection. Zygotes couid also produced by in vitro fertilisation of in vitro matured oocytes.

Fertilised oocytes have two pronuclei: male and female pronuciβuε(PN), which appear 4-8 hours after fertilisation. The developing pronuclei are normally obscured by the htgh content of lipids present In swine embryos. Visualisation of the PN was achieved by ceπtrifugation (6-12 min. B.000-15.000g), Ceπtrifugatioπ stratifies the lipids and allowed both PN to be easily removed by enucieation techniques. Cumulus cells still adhered to the zona pellucida were removed by incubation in hyaluronidaee (300iu mi) for approximately five minutes.

Cytoplasts for the purpose of serial nuclear transfer were prepared from zygoteβ 44-50 hours after hCG (Chorulon, Intervet (Aust) Pty. Ltd.) administration. Fertilised oocyte© 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, Sigma-Aldrich Pty. Ltd.. 300 lU/ml in H-G1 medium or other short term handling medium) and stained in UV fluorescent DNA dye Hσechst 33342 (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd., 5 μg/ml) for approximately 10 minutes at 39^βC. Metaphase ll oocytes were selected . The zone pellucida was partially dissolved by α short inαubation in 0.1% pronaβe (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.) and removed by a gentle passage through a narrow pippette. The oocytes were incubated 15 min in H-G1 or other short term medium with cytochalsln B (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.) (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 enuoleαted halves, identified under UV illumination by the absence of chromatin stained with Hoechst (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.), 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

Karyopiast preparation

Karyopiasts from the following sourceβ were Used: 4- to 16-cell embryos (embryonic nuclei), embryonic foetal, and adult (sematic) 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. Superovuiation was induced as described in Example 1. Foetuses for PGC isolation were collected from 25 to 35 day pregnant female mlnlswine following superovuiation 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 biastomere. 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, Sigma-Aldrich Pry. Ltd.)) 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 πuoleus 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 polymerisation inhibitor, for example nocodazole (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd., 1 μg/ml) (Kwon and Kono, 1996). Alternatively the whole blastomeres, synchronised in the G2 or M- phase were used as the source of the karyoplasts. The ceil cycle consists of four phases: M (mitosis), GO. S (synthesis) and G2. Synchronisation 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 miniβwine, 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 genotypiπg (by using PCR). The fibroblast cultures were established from lung, muscie/skin (meseπchymal) tissues as follows. Other tissues may also be used. The tissue from individual foetuses was disected, washed in PBS (Oxoid, Unipath Ltd. UK), disaggregated mechanically by mincing with scissors or/and enzymatically by incubating for 5 min at 37°C in 0.25% trypsin / 0.04 0 EDTA (Gibco BRL, Life Technologies inc. US) in PBS (Oxoid. Unipath Ltd. UK). Disaggregated cells were washed in culture medium and plated onto gelatinised 10 cm Petrie dishes, at 106 - 107 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 105 -106 cells onto a 10 cm gelatinised 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, Life Technologies Inc., US) supplemented with 10% FCS, 2 mM glutamine (Gibco BRL, Life Technologies inc. US), 100 lU/rnl penioillin (Giboo BRL, Life Technologies inc, US), 100 μg stroptomyoin (Giboo BRL, Life Technologies inc. US), 0.1 mM β mercapto ethanol (Gibco BRL, Life Technologies inc. US). Other types of media or protein/growth factors may also be used (e.g. DMEM (Gibco BRL, Life Technologies Inc., US):F12 (Gibco BRL, Life Technologies Inc. US)) If they are found to provide better culture conditions. The cells were karyotyped al 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 slem ceils isolated from ICM, ED. PGC and EC.

PBS

Sterile solution of PBS (Oxoid, Unipath Ltd. UK), (Ca and g" free) supplemented with 1 00 lU/ml penicillin, 100 μg/ml streptomycin. Culture media

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

Gelatin

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

Trypsin

:0.25% trypsin (Gibco BRL. Life Technologies inc. US), 0.04% EDTA (Gibco BRL Life Technologies Inc. US) In PBS (Oxoid, Unipath Ltd. UK), (without Pen/Strep).

Freezing

In 90% culture medium, 10% DMSO (Sigma Cell Culture, Sigma-Aldrich

Pty. Ltd.).

EXAMPLE 3

Isolation and genetic modifioation of bovine ombryonio fibroblaβtθ

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 lU/mi 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 disected 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 tiεεuee were incubated for 90 min (or up to 4 - 6 hr) at 4^βC 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 dissagregated by vigorous pipetting. Large, undigested pieces of tissue were allowed to settle by sedimentation. Supernatant containing single cells was decanted and Ihe cells plated at 3 x 10⁴ ceils/cm² onto gelatinised (0.1% gelatin in water) Petrie dishes and cultured at 37*C in an atmosphere of 5% CO_∑ in air. This was passage 0 (PO). The medium was ohanged even 24 hours and tho oclls oulturcd until confluent (between 2 to 5 days). The celis were then expanded by trypsinizatioπ and passaging 10^s - 10° cells onto a 10 cm gelatinised 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 g" free) supplemented with 100 lU/mi penicillin, 100 μg/ml streptomycin

culture medium Dulbecco's modified Eagle's medium supplemented with

15% PCS, lOO 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 n PBS (without Pen Strep)

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

The sex of the isolated fibroblaεtε was determined by PCR (Pomp et al J, Anim Sci 1995; 73: 1408-1415). Two genes were amplified in a singie reaction:

Sry (sex-determining region Y); the presence or absence of this gone 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 fibroblaεtε were uεed 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) 6PLaoZ TINβS-MO linearized at α unique Xho I restriction site (β- galactosidase-neornycin resistant fusion gene), or

b) IRES-Neo linearized at a unique Mlu I restriction zite (neomycin resistance gene).

2 x 10^δ BEF-M 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 Na_eHPO*, 6 mM glucose) containing 10 μg of linearized vcotor. 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 Puiεer II apparatuε (Bio-Rad Laboratories). After 10 mm 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 scicoted for 8 to 14 days.

Following electroporation and selection with Qeneticin, 1B 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 TINβS-MO clones were analysed for the expression pattern of β-galactosldase 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 εwine. The tissue samples were briefly sterilised in 70% ethanol and washed in 3 changes of cold PBS containing lOO IU/ml penicillin, 100 μg streptomycin (PBS- PIS). 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^eC in 0.25% trypsin / 0.04% EDTA in PBS. After removing the excess of trypsin, tissues were incubated at 37^ΛC for 30 min, culture medium was added and cells dissagregated by vigorous pipeting. Large, undigested pieces of tissue- were allowed to settle by εedimentation. Supernatant containing single cells was decanted and the ceils plated onto gelatinised (0.1% gelatin in water) Petrie dishes and cultured at 37^υC in an atmosphere of 5% CO₂ 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 10° cells onto a 10 cm gelatinised Petrie dish in culture medium. Aliquots of oelle 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 lU/ml penicillin, 100 μg streptomycin, 0.1 mM β mercaptoethanol

PBS εteriie εolution of PBS (Ca" and Mg^"'" free) εuppiβmented with 100 lU/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, Q.04% EDTA n PBS (without Pen/Strep)

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

EXAMPLE 5

Gene ooπstruots and traπsgenesis

The genetic modification of animal genomes can be broadly divided into random and targeted transgene integration. Whiie 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 ceils, 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 geπelic manipulation with nuclear transfer. Nuclear transfer differs from ES cell-mediated transgenesis in that a variety of cell types that can be cultured and genctioally manipulated in vitro can be utilised 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 germllne of an animal. However, the nuclei of genetically modified embryonic fibroblasts can be utilised as a genetic resource to establish a transgenic animal by reprogramming the developmental capability of that nucleus through nuolear transfer. Conversely, fertilised oocytes or eingle 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 introduoe specific targeted mutations in the germiine of inbred MHC (SLA) miniature swine, we have engineered MHC (SLA) miniature swine genomic libraries and cioned and engineered a range of gene targeting vectors as follows. Figure 2 summarises the steps laken 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 myoεtatin gene to increase muscle growth, 3) Growth modification: directing modified growth hormone conεtruct 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 traπsgeπes to increase disease resistance, interferon gene,

6) Production of human pharmaceutieale,

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 α d/d miniswiπc shown to be homozygoue at the α1 ,3-galactoeyltransfera6e locus by RFLP analysis was used to conetruct 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, KW261 , The genomio library contained approximately 2 x 10⁶ independent donee.

Construction of vector, pBert

The unamplified library was screened with a 240 bp PCR fragment corresponding to nucleotides 607 througn 847 of porcine α1 ,3-galactosyltransferase gene (GT, Strahan et al. 1995.- immunogeπetics, vol. 41 : 101-105, cDNA Sequence and Chromosome Localization of swine αl ,3-gaiactosyitraπsferase, Geπbank Accession Number L36152) using standard methodologies. Six bona tide porcine αl ,3-galactosyitransferase 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- 0 (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 θ 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 oligonucieotides 5'-TCG ACT GTT TAA ACG GCC TCT ACG GCC TAG CT-3'

<SEQ ID NO:1) and 5'-AGG CCG TAG AGG CCG TTT AAA CAG-3' (SEQ ID

NO:2)) as εhown in Figure 4 with cohesive Sal 1 and Sac I ends, which contained a unique Pme I and Sfi i was ligated to the fragment, followed by ligation to pUCl9

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 done. Stop codons were introduced into the 5¹ end of exon 9 by modifying 4-2. The piasmid, 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, daphosphorylated, and ligated together with the 4-2A Eco Rl Sao I fragment. Tho ligatin reaotion was first digested with Pme I, then with Sac I, and the 9-10 kb Pme l Sac I fragment was purified. The vector, pTCR4-9, was digested with Not I, daphosphorylated, 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.etuffer, 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 l Not I fragment isolated.

The four purified fragments: 6.5 kb SuperCos ori (Eco RIVXba I; 12.6 kb SuperCos:stuffer Xba l/Not I; 9-10 kb TCL12A/4-2A Pme l/Sac I; and 11 kb TCR4-9 Sac l/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 wilh 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 II site of pOCUS. A partial Spe I digest was carried out on pOCUS/pgk-πeo, the linearized Spe I vector punfied, then digested with Xho I. The resulting 3.7 kb fragment containing the vector was purified and ligated to the 6.5 kb (Eco RlVXho 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 QT region (with stop codon modifications) from the vector backbone. The unpurified 18.1 kb Eco Rl/Not I fragment was ligated to the pOCUS/pgk-neo Eco Rl/Not l vector. The ligated DNA was electroporated into competent E.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 immortalised to permit sufficient paεεage between the initial transfection and subsequent analyses. Third, the line should express the α1 ,3-galactoεyltranεferaεe gene, as expression will be required for the homologous recombination assay. Fourth, the line must be transfectablε and selectable at reasonable frequencies. Fifth, to facilitate generation of an rel ,3-galactosyitransfernse null line the line should be diploid.

Aortic endothelial cells from the same miniature swine used to derive the genomic DNA library were Immortalised with SV40 T antigen (Seebach et al., 1997, The 4th intemationai Congress for Xenotransplantatioπ, Nantes, France). One of the ceil lines, PΞDSV15, which satisfied the criteria listed above, was transfected by electroporation with the pGallaway targeting vector. The vedor was linearized at a unique Xho i restriction site. 1 x 107 .PEDSV15 ceils were suspended in 0.8 ml of HβBS (20 mM HEPES-NaOH pH 7.05, 137 mM NaC1 , 5 mM KC1 , 0.7 mM Na.HPO*. 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 uF charge was then applied with a Gene Puiser 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 (M1G9 containing 10% fetal bovine eerum (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 10* cell/cm' 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 stabie ciones/plαtθ. The oells were re-fed enhanced medium containing 400 μg/ml Gcnctioin (Life Technologies, Gaithersburg, MD) and εelected for 10-14 days.

RT-PCR Assay or Homologous Recombination

The genomic organisation of the α1 ,3-galactosyltranaferase gene following a homologous recombination event between the chromosomal locus and the pGallaway vector is shown in Figure 6. The length of the u1 ,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 Ihe generation of RNA transcripts containing Ihe mutations

(stop .codons) introduced into the vector. These transcripts are most readiiy identified by RT-PCR using a forward primer from an α1 ,3-galactosyltraπsferase 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 dsπved from the mutated region of exon 9 In the pGallaway vector, and has a 3' terminal mismatch to the wiid-type α! ,3-gaiactosyitransferase 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 Prβamplification System (Life Technologies, Gaithersburg, MD), 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 KCI, 10 μM each dNTP. 0.9 mM MgCI₂. and 25 μ/ml Amptitaq Gold polymerase '(Perkln-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-Elmβr, Norwalk, CT) with the following profile: 3) 95°C for 9 min.

2) 39 cycies of:

96^βC for 2 see 60^βC for 30 sec 72°C for 30 sec

3) 72^βC for 5 min.

Analysis of Targeting Efficiency

Two weeks following electroporation and selection with Geπetlcln aliquots of the stable transfectant pools were analysed 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'-AQA TGA CTT TGT GGC CAA CC-3' (SEQ ID NO:5), from porcine α1 ,3-galactosyltransferase exons 7 and 9 respectively.

To ensure that the RNA poois were not contaminated with mutant template. the mock cDNA reactions were amplified with primers F23B and RD2. To identify poois containing cells producing the mutant transcript expected from the homologous recombination of pGallaway and the endogenous αl ,3-galactosyltransferase locus, the cDNA reactions were amplified with pπmers F238 and RD2.

Initial analysis of 7 pools of approximately 500 stablo 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 bromido staining of PCR products generated from cDNA with the wild type F238 and R545 primers showed that amplifiabie cDNA was produced from each pool.

In panel B. the pool cDNA preparations were amplified with primers F238 and RD2, for specific detediαn of product generated from the expected mutant transcript. Hybridization of Southern blots with an internal α1.3-galactosyltraπsferase 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 analysed.

To eliminate any possibility that the PCR produd 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 oleave 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 amplificalion 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 transcriots expected from a homologous recombination event, for a homologous recombination frequency of 1-2%. ^•To select for a cell line totally devoid of 1 ,3-galadoεyltranεferaee activity, ie., a null cell line, we reasoned that screening the pools of clones for lack of binding to antibodies specific for the Galαl ,3Gal epitope, synthesised by α1 ,3-galactosyltransferase might result in selection ot 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 lyβie. 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 aliele. Small amounts of PCR product derived from the wild type RNA are oompatible either with a small surviving sub-population of GT expressing ceils or with a significant, but not total, lose of wild type RNA in the "null population". A FACS analysis was performed on the 2*3 line following 7 rounds of XNA/comptemβnt selection, and the parental PEDSV15 line to determine the relative levels of binding to Synsorb 90 or to the lectlnlB4. 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 α1 , 3-galactosyltransferase activity.

Having demonstrated that gene targeting of the αl ,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 bul not limited to cultured cells, eg., embryoniα fibroblasts, or embryonic stem cells, or embryos.

The introduction of new genetic material and the subsequent selection of cells harbouring 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 ceils suitable for in vitro genetic modification and subsequent nuclear transfer have limited in vitro propagation capacity. It Is therefore desirable to utilise transfection and selection systems which generate and/or Identify correctly targeted clones at high efficiency and with limited requirement for in vitro propagation,

A particuiariy efficient approach to selecting for correctly targeted clones Is to use* IRES gene trap targeting vootore. 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 πon-tranεcribed regions of the genome are not seiected.

Widely expressed genes, such as the αi ,3-galactosyltransferase gene which is the principal target for gene deletion in xεnotransplaπtation, is well suited to IRES gene trap targeting strategies in nuclear transfer programs. Figure 10 shows^' one α1 ,3-gaiadosyltransferase IRES gene trap targeting vector for f unotiqnαl disruption of α1 ,3-galadosyltransf erase gene expression, in a preferred embodiment, it may be deeirablo to remove the selectable marker cassette from the targeted locus to eliminate expreεεion of the antibiotio resistance gene. One approach Is to flank the IRES selectable marker cassette with εuitable 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 α recombinase sensitive α1 ,3-galactosyltransferase IRES gene trap targeting vedor for fundional disruption of α1 ,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 uncnaracterised transgenic cells should generate offspring of the desired phenotype at a ratio equivalent to the selected pool. The elimination of cloπal 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 bom offspring by nuclear transfer, however, totipotentiai 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 ceils or embryos by direct injection, ciedroporation, 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 ceils. Typically, plasmid DNA for electroporation was linearised by restriction enzyme digest, ethanol precipitated and resuspended at 1.0 mg/ml in PBS (Oxoid, Unipath Ltd. UK). Following 2 hours culture in fresh medium, near confluent cells were dispersed by trypsinisation, washed sequentially in culture medium and PBS (Oxoid, Unipath Ltd. UK), and resuspended at 1.4x108/mi in PBS (Oxoid, Unipath Ltd. UK) for immediate transfedion. Routinely, 0.7 ml of ceil suspension was mixed with 0,1 ml DNA containing solution and electroporated at 0.8 kV and 3.0 mFD using a Biorad Gene Puiser and 0.4 cm cuvettes. Transfections were plated on gelatinised tissue culture dishes at 5-8x104/cm2 In growth medium for 16 hours prior to the addition of selection medium containing 200 mg/ml (active) G418 (Sigma). Single colonies were pioked 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 Ω418 and subsequently in media without G418.

EXAMPLE 6

Elimination of α1 ,3-galactosyltransferase activity in porcine foetal fibroblasts

Mesenchymal tissue derived fibroblasts were used to eliminate α1.3- gaiactosyitranεferaee activity, The cell line, PEM, was transfected by electroporation with the pGallaway targeting vedor linearized at a unique Xho I restriction site. 1 • 4.8 x 10* 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 Na₂HPO₄, 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 200 V, 960 μF ohαrge was then applied with a Gene Pulser II apparatus (Blo-Rad Laboratories). After 10 min incubation at room temperature the cell suspension was transferred to 10 mi of culture medium and plated onto a number of 10 cm Petrl dishes. The cells were re-ted culture medium containing 400 μg/ml Geneticin (Life Technologies) and selected for 8 to 14 days.

Following eledroporation and selection with Geneticin aliquots of the stable transfectants were analysed 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 α1 ,3-gaiactosyltranεferaεe locus.

In two electroporation experiments 7.6 x 10⁶ were transfected and 178

Geneticin resistant colonies isolated and analysed for the elimination of one copy of the αl ,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 harbouring targeted Integrations. To prevent extensive culture of targeted colonies a "master" plate is stored frozen, while duplicate piate(e) are expanded and progressively smaller pools of colonies analysed by RT-PCR for the targeted evenl.

The "master" plate is frozen as follows: When tne cells reach confiuency, 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. 80% FCS) added to each well. The plate Is stored for up to 2 to 4 weeks at -75*0 or after 24 hr al -75'C transferred to liquid nitrogen. To thaw the cells, the plate is transferred to room temperature, 500 μi of warm culture medium added to each well and the cell suspension transferred to a new 24 well plate. Tho cells are cultured at 37°C in an atmosphere of 5% C0₂ in air.

EXAMPLE 7

Nuclear Transfer

Activaition

Activation of the cytoplast was preferred for further embryonic development. Activation occurs during fertilisation and is characterised by a scries of Ca²ⁱ oscillations, release of cortical granules, extrusion of a second polar body, pronuclear formation and ultimately cleavage. Development of an oocyte without fertilisation by sperm is known as parthenogeπic activation. Parthenogenlc ootivation can be induced by various chemical and physical treatments such as ethanoi, calcium ionophore and electrical pulses (Ozil, J.P. 1990). In the swine, electrical puises have been shown to be more effective at indudion of parthenogenetlc development than ethanol (Saito et al., 1993). Electrical stimulation is therefore the preferred method of activation Induction. Oocytes and reconstituted cytoplasts were adivated by exposure to one υr a series of electrical puises (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 biastocyst 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 cytopiaεt with a karyoplast containing tho donor nucleus. Cytoplasts were reconstituted by inducing membrane fusion with the karyopiaεt following treatment with chemical reagents, such as polyethylenegiyool (Czolowska et a!., 1984), or by exposure to electrical. pulses (Wllladsen 1986). Cytoplasts were also reconstituted by direct injedioπ of the intact donor nucleus and karyoplast lysate Into the cytoplast (Bromhall 1Θ75; lllmensee and Hoppe 1981).

.Electrofusion was the preferred technique for cytoplast recoπstitulion. The parameters required for successful fusion depends on the origin of the karyoplast and also whether the zonae pellucida wae present on the cytoplast. When cytoplasts with an intact zonae pellucida were used, the karyoplast was inserted into the perivltelllπe space (PV) of the cytoplast between the oocyte membrane and zone pellucida, with all manipulation performed in cytochalasin B (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd., 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 mannito! (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.), (Prather et al., 1989). For effective fusion to occur, the cytopiast 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 1.0 s) was used to assist in orientation. When aligned, fusion was induced by a brief exposure to DC puise (30-120 V/mm for 50 - 300 μs). Fusion of the oytoplast and karyoplast was observed within 15 -30 minutes by loss of membrane distinction between cells.

iCytopiast volume has been demonstrated to effed development of nuclear transfer embryos (Peura et al 1997). It may thorefore 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 iεofuεion 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., 1987) and cattle (Cibelii 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 enucieation, 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 NIT embryos

-

"WT" : wild type nuclei donor

"KC^* : modified nuclei donor

Only reconstituted MT porche embryos have been transferred to recipients.

While a published nuclear transfer approach has produced live onspππg in cattle (Clbelll 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 nuciear 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 (aee Table 7 and Example 2). TABLE 7

Somatic Cell (Adult Cell) Nuclear Tranefer in the Pig

Universal donor

The nuclear tranefer 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 poroine Nuoleβr Tranefer

Serial nuclear transfer

Alternatively, the aotivated reconstituted oocytes may be allowed to undergo one round of karyokinβsis. 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 Borardino 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 cytochaiasin B (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.. 5 bagel) at 39^βC in 5 0 CO_z for 6 hours (Kwon and Kono 199Θ). Following activation the donor nucleus re-βntcrod the ceil cycle and completed karyokinesis. The addition of cytochaiasin B (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd.) 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 fertilised oocyte (aee 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 tranefer, and therefore increase pregnancy rate, nuclear transfer may be performed using karyoplasts from 4-cell nuclear tranefer embryos. In this example, the nuclear transfer procedure was used as a means of embryo multiplication. Aε 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 θlectrofusion 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, Sigma-Aldrich Pty. Ltd., 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 iung (PEL) fibroblasts

• pig embryonic mesβπchymal (PEM) fibroblasts • pig embryonic heart (PEH) fibroblasts

• pig embryonic kidney (PEK) fibroblasts.

Propagation

Examples of all tour 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 oell 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 ie 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 optimised 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 αl ,3-galactαsy!transfera8e activity in porclno embryonic fibroblasts

Porcine embryonic mesenchymal tissue derived fibroblasts were uβed to confirm the feasibility of elimination of α1 ,3-galactosyitraπsfcrasQ activity. The cell line, PEM, was transferred by electroporation with the pGallaway targeting bector linearized at a unique Xho I restriction site as described in Example 3. 1 x 0° PEM cells were suspended In 0.4 ml of HeBS (20 M HEPES- NaOH pH 7.05, .137 mM NaCl, 5 mM KCI, 0.7 M Na_∑HPO*, 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 270 V. 960 uF 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 plaled onto 10 cm Petri dish. The cells were re-fed culture medium containing 400 μg ml Geneticin (Life Technologies) and eeiected for 8 - 14 days.

Following electroporation and selection with Geneticin aliquots of the stable transfectants were analysed by RT-PCR as described in Example 3, Two sets of primers were used: 1) wild type (primerε F238 and R545) to verify that cDNA was transcribed successfully;

2) mutant (primers F238 and RD2) to identify the oolis produoing the mutant transcript expected from the homologous recombination of pGallaway and the endogenous α1,3-galactosyltransferase 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 Iri 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 Ihe same s ie. However, cleavage treatment with Ase I yields two fragments of 272 bp and 24 bp in the mutant PCR product but no oleαvage 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 αf Embryos

Reconstituted oocytes were cultured in 20 μ.l drops ot medium overlaid with paraffin oil (BDH Laboratory Supplies, UK or Sigma Chemical Company, Sigma-Aldrich Pty. Ltd.) under 5% C02 and reduced 02 (5-7%) atmosphere at

39° C. G1 medium was used for the first 48 hr. followed by G2 medium. The combination of G1 and G2 medium and the reduced 02 greatly improved the quality of the early embryo and increased the nurnoer 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, Sigma-Aldrich Pty. Ltd. or Gibco BRL, Life Technologies inc.) buffered G1 medium with 4 mg/ml BSA (Miles Pentex or Gibco BRL, Life Technologies inc.)

2) Hepes (Sigma Cell Culture, Sigma-Aldrich Pty. Ltd. or Gibco BRL, Life technologies inc.) buffered MEM with 5 mg/ml BSA (Miles Pentex or Gibco BRL, Life Technologies inc.). The following long term culture media were used:

1) Whitteπs medium with 15 mg/ml BSA (Miles Pentex or Gibco BRL,

Life Technologies Inc.)

2) G1/G2 medium with 4 mg ml BSA (Miles Pentex or Gibco BRL, Life Technologies inc.).

The embryos were cultured in micro drops of medium overlaid with mineral oil (B H Laboratory Supplies, UK or Sigma Chemical Company, Sigma-Aldrich Pty. Ltd.), in 5%CO_a: 5%0₂: 90%N_a atmosphere, at 3R.5^SC. The embryos were cultured for up to 7 days before embryo transfer to suitable recipient female.

The chemical composition of G1 and G2 media is shown below. H-G1 is a hepes . added version of G1 medium. All chemicals are Sigma, except for BSA (Gibco, or Miles Peπtax).

G1 from concentrated stocks

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

Stock 10 ml 50 ml

AG_I 1.0 ml 5.0 ml

B 1.0 ml 5.0 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

NonΕssential AA (x100) 0.1 ml 0.5 ml

H₂0 7.4 ml 37.0 ml

BSA 30 g^" 150 mg

H-G1 from concentrated stocks

Each reagent was added to MQ H₂O 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

Noπ-Essential AA (X100) 0.1 ml 0.2 ml 0.5 ml

HEPES 0.84 ml 1.68 ml 4.2 ml

HaO 7.4 ml 14.8 ml 37.0 ml

BSA 30 mg 60 mg 150 mg G1 & H-G1 PREPARATION:

Preparation of Concentrated Stocks:

Concentrated stocks were prepared by dissolving components In sterile MQ H20. Each stock was filtered and stored at 4^βC for 3 months (Stock AQ∑). 1 month (Stook D) or one week (Stocks B. DGZ and G).

Stock AQI Component mM g/100ml

(x10 concentration) NaC1 85.16 5.2Θ5

KC1 5.5 0.410

NaH₂P0₄.2H₂O 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/100ml

(x 10:concentratιon NaHCO₃ 25.0 2.101 Phenol Red 0.01*

Stock Coi Component M g/10ml

(x 100 concentration) NaPyruvate 0.32 0.0352

Stock D Component mM g/10ml

(x 100 concentration) CaCI₂.2H_zO 1.8 0.265

Stock G Component mM g/10ml

(x 100 concentration) Glutamine 1.0 0.146

Stock T Component mM g/10m!

(x 100 concentration) Taurine 0.1 0.0125 Stock ED Component mM g/lOml

(x 100 concentration) EDTA 0.1 0.029"

Stock HEPES Component mM 9710ml

(x 100 concentration) HEPES 20 5.958^c Phenol Red 0.01"

Usually only 0.0019 added ^b EDTA must be dissolved In 220 μl 1 M NaOH prior to adding HaO ^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 HgO. Each stock was Altered and stored at 4°C for 3 months (Stock A_M), 1 month (Stoc D) or one week (Stocks B. Doe and G).

Stook AQ_I Component mM g/100ml

(x10 concentration) NaC1 85 16 5.265

KCI 5.5 0.410

NaH₂P0₄.2H_aO 0.5 0.078

MgSOi 7H?0 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/IOOml

(x 10 concentration) NaHCOs 25.0 2.101

Phenol Red 001*

Stook C_β2 Component mM g/10ml

(x 100 concentration) NaPyruvate O 10 O 011

Stock D Component mM g/10ml

(x 100 concentration) CaCI₂.2H_a0 1.8 0.265

Stock G Component mM g/lθml

(x 100 concentration) Glutamine 1.0 0.146

Usually only 0.001 g 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 1 M NaOH/10ml). The medium was filtered and stored at 4°C. Osmoiaπty should be 269 mOsm.

Stock 10 ml 50 ml _a* 1.0 ml 5.0 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-Eseential AA (x100) 0.1 ml 0.5 ml

Essential AA (x50) 0.2 ml 0.6 ml

H_sO 7.4 ml 37.0 ml

BSA 30 mg 150 mg

EXAMPLE 10

Generation ot transgenic Offspring by Nuclear Transfer

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

To demonstrate the generation of transgenic animals from nuclear transfer we have developed a novel approach using a mouse moαel. This approach involves the use of a unique transgenic mouse that had a nuclear localised lacZ-neoR expression as a result of random gsπe-trap Integration of pGTIRES-βgeopA into what appears to be an ubiquitously expressed gene. As a result all ceils 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 usod in nuclear transfer. Blastocysts that developed following in vitro culture ot 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 bom following blastocyst tranefer aiεo displayed distinctively blue staining (Figure 12.2 nuoloar transfer pups with litter mates and

Figure 12.3 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 strain of swine with a genetically defined major hlstocompatlblllty 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 produoed by nuclear transfer. EXAMPLE 12

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

Oestrous Synchronization

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

Method 1: Corpus luteum regression following Prostaglandin treatment

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

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, Pltmaπ-Moore Australia Ltd.) or

Prostaglandin F2u analogue followed by 500 IU 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 asm..*? (standing heat)

Day 7 One ceil 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 wilh Altrenogest (Regumate. Roussel-Ulcaf, Paris, France) for 14-1 B days (15-20 mg/animal day) to synchronise estrus cyolcs. Twonty four hours after removal of Regumate (Altrenogest, Hoechst-Roussel) swine were injected with 500 lU of PMSG (Folligon, Intervet (Aust) Pty. Ltd. or Pregnecol, Horizon Technology Pty. Ltd.) followed two days later Injection of 500 lU of hCG (Chorulon, intervet (Aust) Pty. Ltd.) .

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

Recipients were synchronised for embryo transfer at weaning ueing 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.) 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 1 1 Blastocyst transfer.

Surgery Standard surgical procedures were used. Following induction of anaesthesia with Thiopentone, surgical anaesthesia was maintained with either halσthane (fluothane, Zeneca. ICI Australia Operations Pty. Ltd.) or Isofluorane (Forthane, Abbott Labs, UK) in oxygen. Reproductive tracts were exteriorised by midlinc iaparotomy.

Oocytes or pronuciear stage embryos were collected through a modified teflon catheter inserted into the oviduct via the infuπdibullum. Embryos were collected by retrograde flushing with flushing medium (Dulbecco's phosphate buffered saline (Gibco BRL, Life Technologies inc., NY or Trace Bioscienceε 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 synchronised 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, K 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.

References

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2) Campbell K. H. S._t McWhir, J., Ritchie, W.A.,and Wilmut, I. (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature 3B0, 64-66.

3) Cheong, H«T., Takahashi, Y„ and Kanagawa. H. (1993) Birth of mice after transplantation of early cell-cycle stage embryonic nuclei into enucleated oocytes. Biol. Rβprod. 48, Θ58-Θ63.

4) Cibelli, J.B., Shee, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C, Ponce de Leon, F.A. and Robl, J.M. (1998) Cloned Transgenic Calves produced from Nonquieβcent Fetal Fibroblasts. Sciβnco 280: 2256 - 1258.

5) Czowlowka, R., Modlinski, J.A., and Tarkowski, A. K. (1984) Behaviour of thymocyte nuclei in non-aotivated and activated mouse oocytes. J. Cell Sci. '69, 19-34.

6) OiBerardino, M.A. (1987) Genomic potential of differentiated ceils analysed by nuclear transplantation. Am. Zool. 27, 623-644.

7) Funahashi, H., Cantley, T., Stumpf, T.T., Teriouw, S.L. and Day. B.N. (1994a) Developmental ability of porcine coyotes matured and fertilised in vitro Thoriogβnology 41 :1425-1433.

8) Funahashi, H,, Cantley, T.C., Stumpf, T.T., Teriouw, S.L. and Day, B.N. (1994b) Uss of Low-Salt Culture Medium for in Vitro Maturation of Porcine Oocytes is Associated with Elevated Oocyte Glutathione Levels and Enhanced Male pronuclβar Formation after in Vitro Fertilization. Biology of Reproduction 51 :633-639.

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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 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.

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 72 wherein the couplet is subsequently subjected to a cell fiision/activation step.

5. A method according to Claims 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 Claims 2, 64 or 65 1 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 s 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 ╬▒l,3-galactosyltransferase gene.

21. A method according to Claim 20 wherein the DNA construct is a plasmid selected from the group consisting of pGallaway, pBertl and pBertll, 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 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 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 Claims 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 Claims 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 ╬▒l,3-galactosyltransferase gene.

43. A method according to Claim 42 wherein the DNA construct is a plasmid selected from the group consisting of pGallaway, pBertl and pBertll or a functionally equivalent plasmid.

44. A method according to Claim 34 or claim 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 produced by a method according to Claims 24, 25 or 26.

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

47. A transgenic 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 transgenic animal according to claim 48.

51. An organ derived from a transgenic 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 construct according to Claim 54 wherein the animal nucleus is a swine nucleus and the endogenous gene is* ΓÇó 1,3-galactosyltransferase.

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 ΓÇó 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 construct according to Claim 60 wherein the animal nucleus is a swine nucleus and the endogenous gene is ΓÇó 1,3-galactosyltransferase.

62. A cell or cell line containing a DNA construct 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 Claims 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 Claims 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 transgenic animal produced by a method according to Claims 64, 65 or 67.

69. A transgenic animal descended from a transgenic 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.