A background to nuclear transfer and its applications in agriculture and human therapeutic medicine

Abstract

The development of a single celled fertilized zygote to an animal capable of reproduction involves not only cell division but the differentiation or specialization to numerous cell types forming each tissue and organ of the adult animal. The technique of nuclear transfer allows the reconstruction of an embryo by the transfer of genetic material from a single donor cell, to an unfertilized egg from which the genetic material has been removed. Successful development of live offspring from such embryos demonstrates that the differentiated state of the donor nucleus is not fixed and can be reprogrammed by the egg cytoplasm to control embryo and fetal development. Nuclear transfer has many applications in agriculture and human medicine. This article will review some of the factors associated with the success of embryo development following nuclear transfer and outline the potential uses of the technology.

Introduction

In animals reproduction occurs by sexual means; fertilization of the female egg by the male-derived sperm results in the production of a single cell or zygote, which begins development and results in the production of offspring. The genome or genetic information present in the majority of the cells of the body consists of two sets of genes, one contributed by the sperm (the paternal genome) and the other by the egg (the maternal genome). During the development of egg and sperm cells the genetic information is rearranged by the process of meiosis, and thus each fertilized zygote contains a unique genome and results in the formation of a unique individual. During development the zygote has to grow and divide; with each division the genome is copied and each cell inherits a copy of this novel genome which is located within the nucleus (also referred to as nuclear or chromosomal DNA). In contrast, a clone is defined as a population of cells or organisms derived from a single cell or organism through the process of asexual reproduction. Under natural circumstances, the occurrence of ‘clones’ in vertebrates is restricted to the production of identical twins as a result of the division of a single embryo to form two identical but separate individuals (also termed monozygotic, derived from a single zygote). In some species division of the embryo may result in the formation of more than two clones (e.g. in the Echinida). Such ‘splitting’ of embryos may also be carried out experimentally in the laboratory to produce identical offspring.

In contrast to the ‘splitting’ of embryos, Dolly was produced by a technique known as nuclear transfer. In this process the genetic material is removed from an unfertilized egg and replaced with the nuclear genetic material taken from a cell at a later developmental stage or, as in the case of Dolly, a cell derived from an adult animal. No fertilization occurs and therefore there is neither maternal nor paternal DNA present in the resulting zygote. In normal sexual reproduction the egg and the sperm are not equal contributors; although both contribute genetic information, the egg contains numerous other factors essential for development. In particular, the egg contains extrachromosomal DNA located in the mitochondria; these intracellular organelles are inherited primarily through the maternal line. In the true sense of the word the animals produced by nuclear transfer are not ‘clones’ as eggs used for the process are obtained from different females and therefore maternally inherited factors (i.e. mitochondria) will differ between the resultant offspring. Although the offspring are generated by asexual means, cell duplication or splitting did not occur and therefore may be more aptly described as ‘genomic copies’.

Background to the development of ‘cloning’

As described, the product of sexual reproduction the zygote inherits a single maternal and a single paternal copy of the genome. From this combined genetic material and the maternally inherited organelles, proteins, mRNAs, etc., found in the unfertilized egg, an embryo, fetus and finally an adult animal develop. The single-celled zygote must grow and multiply to produce the vast number of cells which make up an individual, and in addition groups of cells must develop along specific pathways in order to produce all of the cell types which make up the tissues and organs of the mature adult. The controls of the processes which determine this specialization, termed differentiation, form one of the most fundamental questions in developmental biology. Today we understand that genetic information is encoded by DNA, which is assembled into chromatin and makes up the chromosomes. However, although early developmental biologists realized that the zygote must contain all of the information required to produce a complete individual, the nature and control of this information was unknown.

Two theories arose to explain the control of differentiation; August Weismann (1892) proposed that during development the genetic material was ‘shared out’, i.e. individual cells received only that information which was required to fulfil their particular specialized function. Credence to this theory was lent by experiments using sea-urchin embryos. Wilhelm Roux (1888) killed a single blastomere at the two-cell stage by puncturing it with a hot needle; following further development a half embryo was produced.

However, in subsequent experiments, Driesch (1892) and others separated the blastomeres at the two-cell stage and found that anatomically whole, but dwarfed embryos were produced (for a comprehensive history see Di Berardino, 1997). These experiments demonstrated that at least during early embryonic development, each cell retains all of the genetic material. In an extension of these experiments, Hans Spemann constricted salamander zygotes, using human baby hair, restricting the nuclei to one half of the cytoplasm. The half zygote containing the genetic material continued to divide. After several divisions the constriction was relaxed sufficiently for a nucleus to traverse the cytoplasmic bridge. The previously enucleated portion of cytoplasm then resumed cleavage and development. This technique resulted in the production of dwarfed but twinned offspring. After considering the results of these experiments, Spemann proposed the transfer of nuclei from more advanced developmental stages back to zygotes from which the genetic material had been removed (Spemann, 1938).

This suggestion by Spemann, which is perceived as the birth of nuclear transfer technology, was originally proposed as a method to study the role of the genetic material in cellular differentiation or, more specifically, whether information contained within the genes was lost or inactivated during development and differentiation. We now know, from many lines of research, that development and cellular differentiation is the result of highly specific temporal and spatial control of gene expression. During these processes DNA is not lost and the majority of cells in an adult animal retain two copies of the genome as originally inherited in the zygote.

A brief history of cloning

Although Spemann provided a route for the investigation of differentiation by nuclear transfer, further experiments were not reported until the 1950s when Briggs & King (1952) reported the development of swimming tadpoles after the transfer of nuclei from early-stage frog embryos into enucleated eggs. Subsequently, John Gurdon reported the production of adult Xenopus (South African Clawed Toads) after transferring nuclei from tadpole intestinal epithelial cells (Gurdon & Uehlinger, 1966); however, in subsequent experiments no adults were produced when cells taken from adult animals were used as nuclear donors (Gurdon et al. 1975). In mammals, the techniques for embryo reconstruction were not developed until much more recently, one reason being the small size of mammalian eggs, 120–150 µm, in comparison with those of various amphibians, which are > 1 mm. Therefore, significantly different equipment is required; however, there were early reports of nuclear transfer in the rabbit during the late 1970s and then McGrath & Solter (1983a) demonstrated that by using microsurgery, pronuclei could be exchanged between mouse zygotes and development still be obtained. Subsequently, Willadsen (1986) produced live lambs after transferring nuclei from 8- to 16-cell sheep embryos into enucleated MII oocytes. Following these reports, successful nuclear transfer was reported in cattle, sheep and mice by a number of laboratories, but development to term was restricted to the use of early cleavage-stage embryos as nuclear donors.

Methodology of nuclear transfer

In mammalian species, enucleated MII oocytes (unfertilized eggs) have now become the recipient cell of choice owing to the lack of development obtained when using enucleated zygotes (i.e. in cattle: Robl et al. 1987; pig: Prather et al. 1989). This restriction may be due to the removal of zygotic factors, which are essential for early development that may be associated with the pronuclei. MII oocytes to be used as nuclear recipients may be obtained from a variety of sources dependent upon species.

In ruminants, particularly cattle, oocytes may be aspirated from ovarian follicles following slaughter and matured in vitro. Additionally, matured oocytes may be flushed from the oviducts of donor animals following superovulation regimes and immature oocytes may be aspirated from follicles in vitro or following ovariectomy (for a review see Campbell & Wilmut, 1994). Having obtained a suitable recipient oocyte, the genetic material located on the meiotic spindle is removed by microsurgery. Briefly, a small amount of cytoplasm is aspirated from directly beneath the first polar body using a fine glass pipette. Fluidity of the cell membranes allows both the oocyte and the aspirated karyoplast to reseal following manipulation. The enucleation procedure can be monitored by staining the aspirated karyoplast with a DNA-specific fluorochrome, e.g. Hoechst 3332 (for methodology see Campbell et al. 1998). Following enucleation, the genetic material from the donor cell (karyoplast) must be introduced into the enucleated recipient cell (cytoplast). In the mouse, Sendai virus was originally used to cause fusion of the two cells (McGrath & Solter, 1983b); however, more recently, the development of piezo-aided injection has proved of considerable use (Wakayama et al. 1998). In farm animal species, the use of a DC electrical pulse (electrofusion) has become the method of choice; although viral and chemical methods have been used, these have proved less reproducible and in the case of chemical fusion may be toxic. At this point the reconstructed embryo is able to begin development, but many factors are involved in the successful development of such reconstructed embryos. These include other techniques associated with the methods of activation (induction of fertilization responses), culture, and biological factors relating to both the cytoplast and the karyoplast (for reviews see Campbell & Wilmut, 1994, 1998). Of great importance for successful development is co-ordination of the nuclear and cytoplasmic cell cycle phases of both donor and recipient cells (for review see Campbell et al. 1996a).

Cell cycle co-ordination in nuclear transfer reconstructed embryos

The cytoplasm of the enucleated oocyte remains arrested at MII in the absence of genetic material until an activation stimulus is applied. In all eucaryotic cells the onset of mitosis and meiosis is controlled by a cytoplasmic kinase activity termed MPF (mitosis/meiosis/maturation promoting factor). MPF kinase activity increases during the G2 phase of the cell cycle and causes dissolution of the nuclear membrane, chromatin condensation and changes in the cytoskeleton. The kinase activity of MPF is maximal at metaphase of the mitotic/meiotic division after which it declines rapidly allowing decondensation of the chromosomes and reformation of the nuclear membrane. In MII oocytes MPF activity remains at high levels; when donor nuclei are transferred into this cytoplasmic environment they respond to the MPF and undergo nuclear envelope breakdown (NEBD) and precocious chromosome condensation (Campbell et al. 1993). As the majority of nuclei would unlikely to be about to enter mitosis at the time of transfer, this has been termed PCC or premature chromosome condensation. The effects of PCC on the donor nucleus are dependent upon its cell cycle stage at the time of transfer. Nuclei in the G1 phase (prior to the DNA synthetic period or S-phase) or in then G2 phase (post S-phase) form single or double chromatids, respectively, and suffer no apparent DNA damage. In contrast, the chromatin of nuclei that are undergoing DNA synthesis (S-phase) has a typical ‘pulverised’ appearance and suffers large amounts of DNA damage.

NEBD due to MPF has a second effect: during a single cell cycle the nucleus must replicate all of its DNA once but only once. Failure to replicate a portion of the DNA or to replicate a portion more than once will lead to aneuploidy in the resultant daughter cells and affect development. Central to the control of DNA replication is the maintenance of an intact nuclear envelope. Thus when nuclei are transferred to MII oocytes with high MPF, NEBD occurs and a DNA replication occurs in all donor nuclei regardless of their cell cycle phase at the time of transfer. Only those nuclei that were diploid or pre S-phase at the time of transfer will give rise to daughter cells of the correct ploidy.

Alternatively, if the MII oocyte is activated and MPF activity has declined prior to cell fusion, then no NEBD or PCC occur. Nuclei from G1, S or G2 stages of the cell cycle undergo co-ordinated replication and give rise to diploid daughter cells. When using donor cells at unknown stages of the cell cycle then the use of such preactivated oocytes or ‘Universal Recipients’ is advocated. In contrast, if nuclei can be selected or maintained in a pre-S, diploid stage then these may be transferred to MII oocytes with high MPF activity (for review see Campbell et al. 1996a).

The cytoskeleton and mitotic spindle can also affect the ploidy of the reconstructed embryo. In the mouse, transfer of G2 phase nuclei to enucleated MII oocytes has been reported to result in the production of a polar body and development of live offspring (Cheong & Kanagawa, 1993). This suggests that an intact spindle is formed and a mitotic or pseudo-mitotic event occurs.

Donor cell cycle effects

When working with early embryonic blastomeres as donors of genetic material, both the cell cycle phase of the nucleus and the developmental stage of the embryo donor have been shown to have an effect upon development. During early embryonic development little or no transcription occurs from the zygotic nucleus. At later stages the zygotic nucleus becomes transcriptionally active and assumes developmental control. In early studies on nuclear transfer there was an apparent association between those species in which zygotic transcription occurs at a later stage and the stage at which successful development can be obtained. In those species where transcription occurs later, development from later developmental stages has been possible. This was interpreted in two ways; firstly that in species where zygotic transcription occurs early, then the donor blastomeres were more differentiated. In contrast, when zygotic transcription occurs later during development the donor blastomeres are less differentiated.

An alternative explanation is that in those species which initiate transcription at a later developmental stage, then following nuclear transfer the donor nucleus is able to undergo more mitotic divisions in the absence of transcription and therefore to interact with maternal factors controlling development to a greater extent. Studies examining development from different donor recipient cell cycle combinations have identified a possible window of opportunity for maximal development within the donor cell cycle. Donor cells transferred in late G2-phase, during M or early G1-phases promote a greater frequency of development. This may be due to changes in the donor chromatin that may result in the dissociation of factors involved with transcription. In turn this may facilitate the association of maternal factors which are important for the temporal and spatial pattern of gene expression required for successful development (Campbell, 1998b).

Nuclear transfer from cultured cell populations

Until 1995 development of mammalian nuclear transfer reconstructed embryos was restricted to the use of donor genetic material from early embryos. Although this technique has a number of applications both on a scientific and a technological level, it was the goal of many scientists to obtain development of nuclear transfer embryos from cells which could be maintained in culture. Many reports suggested that specific ‘pluripotent’ cell types (e.g. embryonic stem cells (ES)) would be required; however, in farm animal species such cell populations have as yet not been identified. In the mouse, development to term of embryos reconstructed using ES cells as nuclear donors has recently been reported, but problems with epigenetic instability occur during in vitro culture (Humpherys et al. 2001).

Previous reports had demonstrated that offspring can be obtained by nuclear transfer using inner cell mass cells of blastocyst-stage embryos in both cattle and sheep (Sims & First, 1994). Rather than try to isolate a ‘pluripotent’ ES-like cell line we followed the ability of blastocyst-derived cells to produce live offspring when placed into culture and used as donors of genetic material for nuclear transfer. Owing to the seasonal nature of ovine reproduction, these experiments were initiated over the winter of 1993–1994. To avoid the need for synchronization of the donor cell cycle, nuclear transfer embryos were reconstructed using pre-activated enucleated MII oocytes as recipients. During early passages (P1–P3) live offspring were obtained; however, on continued culture (P6–P11) and subsequent embryo reconstruction during the winter of 1994–1995, no further offspring were obtained. Immunofluorescent analysis of this cell population demonstrated the presence of A type lamins, cytokeratin and vimentin, which are associated with differentiated cells (Campbell et al. 1996b).

An alternative to selecting a cell type which may be successful at controlling development in nuclear transfer embryos is to modify the donor chromatin structure prior to embryo reconstruction. Previously, this paper has discussed the cell cycle in relation to growing cells: an additional cell cycle phase termed G0 is found in cells which have exited the cell cycle in response to a number of conditions. Such cells are said to be quiescent and the G0 stage has been implicated in cellular differentiation. G0 cells are arrested in a post M pre S-phase state with a diploid DNA content and may therefore be transferred to MII oocytes with high MPF activity.

In our experiments, cells were induced into a quiescent state by serum starvation. Quiescent donor nuclei were transferred to MII oocytes at the time of activation, prior to activation and following activation. Live lambs (five in total) were obtained from all combinations, but unfortunately two of these died within minutes of birth and a third at 10 days following birth with a range of congenital abnormalities. The remaining two lambs remain healthy and both have proved to be fertile (Campbell et al. 1996b).

To confirm and extend these studies they were subsequently repeated using a male day 9 embryo-derived cell population, primary fetal fibroblasts from a day 26 fetus and a mammary epithelial cell line isolated from a 6-year-old ewe. Live offspring were obtained from each of these cell populations, the adult cell giving rise to the birth of ‘Dolly’ (Wilmut et al. 1997). Since this time we have seen a number of reports in cattle, sheep and mouse reporting the birth of live offspring from fetal and adult cells which have been induced to enter a quiescent state, or exist in a quiescent state in vivo.

The potential role of quiescence in successful development of nuclear transfer reconstructed embryos using cultured cell populations is presently unclear. The use of a diploid cell line allows co-ordination of donor and recipient cycles, the use of MII oocytes maximizes the number of mitotic events which the donor nucleus passes through in the absence of transcription, changes in the donor cell including a reduction in transcription, a reduction in translation, active degradation of unnecessary mRNA and chromatin condensation are factors which may facilitate interaction of the donor chromatin with maternal factors in the recipient oocyte cytoplasm.

Today there are a number of independent reports of live offspring produced by nuclear transfer from embryonic, fetal and adult cell cultures in sheep (Wells et al. 1998), cattle (Wells et al. 1999), mice (Wakayama et al. 1998), goats (Baguisi et al. 1999) and pigs (Polejaeva et al. 2000). In many of these reports, primary cell populations have been established in culture. Such populations are not clonally derived and therefore it is difficult to compare or predict the behaviour of the population. In cultured primary cell populations the number of cells able to complete the cell cycle and divide decreases as a function of age in culture. Individual cells will enter a non-growing but viable condition, which has been termed senescence. In vivo, many senescent cells complete DNA replication but do not go on to divide, arresting in the G2 phase of the cell cycle and therefore tetraploid (i.e. liver). During culture in vitro it has been reported that on the way to true senescence (i.e. G2 arrested) individual cells can enter a quiescent or G0 state. In any primary cell population in culture one can hypothesize that at any one time a percentage of the cells will be non-growing and diploid. Whether this is a result of suboptimal growth conditions or cells entering a ‘presenescent’ G0 phase is yet to be determined (Campbell, 1998a).

The only true measure of the efficiency of the NT process is the production of viable offspring. The development of reconstructed embryos is influenced by many factors, including quality of the recipient oocyte, method of activation and culture methods. Similarly, induction and maintenance of pregnancy is dependent upon a range of factors influenced both by the quality of the transferred embryo and the age, seasonality, nutritional and hormonal status of the surrogate recipient. Studies on the optimal cell cycle stage of donor and recipient are underway in many laboratories, but to date no clear answer to the problem has surfaced.

Problems and abnormalities

The development of offspring produced by nuclear transfer is an inefficient process, with the majority of studies reporting between 0.5 and 5.0% development to term. Losses occur throughout gestation, at birth and following birth, and a range of developmental abnormalities have been reported. The reasons for these abnormalities are unknown but may reflect incomplete or inappropriate reprogramming possibly related to problems associated with imprinted genes (Young et al. 2000; Young & Fairburn, 2000). A greater understanding of the control of normal development may help elucidate the mechanisms involved in these processes.

Implications of nuclear transfer from cultured cells

Nuclear transfer using embryonic blastomeres as nuclear donors has a number of applications in agriculture and research for multiplication of elite embryos or for the production of multiple copies for research purposes. However, these applications are limited by the number of donor cells available and the efficiency of the process. The use of cultured cell populations can increase the number of animals which may be produced from an elite embryo, fetus or adult. In addition, the storage of frozen cell populations may prove useful in the preservation of genetic resources in a number of species. However, in the short term, the major implication of the use of cell populations that may be maintained in culture prior to their use as nuclear donors is the provision of a route for the precise genetic modification of farm animal species. Previously transgenic farm animals were produced by pronuclear injection; this route to genetic modification will be discussed in relation to nuclear transfer, subsequently specific applications of genetic modification for human therapeutic use will be cited.

Advantages of nuclear transfer for genetic modification of farm animal species

The addition of genetic material or production of a transgenic animal can be achieved by the injection of the required gene into the pronucleus of a zygote. Although this technique has been applied successfully in a number of species including mice, rabbits, pigs, sheep, goats and cattle (for review see Wall & Seidel, 1992) there are a number of disadvantages. (1) Integration does not always occur during the 1st cell cycle, resulting in the production of mosaic embryos (Burdon & Wall, 1992). (2) Integration occurs at random within the genome resulting in variable expression of the gene product. (3) At present only simple gene additions may be performed. (4) The selection of transgenic embryos prior to their transfer is hampered by mosaicism (Rusconi, 1991). (5) The production of the required phenotype coupled to germ line transmission may require the generation of several transgenic lines. (6) Multiplication of the required phenotype or its dissemination into the population is restricted by breeding programmes.

In contrast, the production of offspring from a single cell or cloned population offers significant advantages. Genetic modification can be performed in culture and the modified cells selected prior to animal production. It will be possible to remove (knockout) as well as to add genes, and precise modification of control regions or addition of genes to specific regions of the genome (knockin) will be facilitated. The production of an animal from a single nucleus removes the problems associated with mosaicism; all of the cells within the resultant animal will contain the modification which will be transmitted through the germ line. All of the animals produced will be transgenic and flock or herd generation can be accelerated by producing multiple copies from the cultured cells. The experiments which led to ‘Dolly’ involved the use of mammary epithelial cells. The use of this cell line was related to the potential screening of transgenic cells for milk production in vitro prior to animal production. Thus it may be possible to predict expression level and select the highest expressing cell populations prior to animal production.

In order to carry out these modifications the cultured cell populations must be amenable to transfection and selection in culture and maintain their ability to be used for successful nuclear transfer. It has been demonstrated that fetal fibroblasts are suitable for this purpose with the production of ‘Polly’, a nuclear transfer lamb transgenic for human factor IX derived from a transfected, selected cell population (Schnieke et al. 1997). These experiments also demonstrated that the efficiency of animal production was increased over two-fold, in terms of total animals used, as compared to pronuclear injection. More recently, live lambs have been produced which carry a deletion of a single allele of the collagen gene (McCreath et al. 2000) as well as targeted addition of a gene to the collagen locus. In addition, fetuses and offspring have been produced with knockouts of the alpha 1–3 galactosyl transferase and PrP genes (Denning et al. 2001).

The generation of animals carrying multiple genetic modifications requires the sequential addition, removal or modification of specific genes. In culture, primary cell populations have a finite lifespan; however, by re-deriving cell populations from embryos, fetuses or offspring produced by nuclear transfer it will be possible to extend the period that cells can be maintained in culture to carry out these modifications.

Uses of genetic modification in human medicine

The ability to carry out precise genetic modification on cells in culture not only provides a method for the improvement of present transgenic technology but also facilitates previously improbable genetic modifications. Transgenic animals can play a role in a range of human therapies these include:

1. Biopharmaceuticals: the production of human proteins in transgenic animals. Human proteins may be produced in a range of tissues and bodily fluids including blood, urine and milk. Although each of these may play a particular role, the value of biopharmaceutical production in transgenic animals lies in the high volume which can potentially be produced at relatively low cost. To this end, the production of proteins in the milk of sheep, goats and cattle provides a useful route, although for products required in small amounts transgenic rabbits may also be used. A range of therapeutic proteins are being produced in the milk of transgenic animals including alpha-1-antitrypsin (for treatment of cystic fibrosis) factor IX (for treatment of haemophilia B) (for review see Garner & Colman, 1998). Nuclear transfer will facilitate the removal of endogenous genes to aid purification, for example the replacement of bovine serum albumin with human serum albumin (HAS) in order to produce large amounts of HSA for the treatment of burns.

2. Nutraceuticals: in contrast to the production of proteins for treatment of specific medical disorders, nutraceuticals have been defined as products which may have medical benefits but are not specifically treatments. Examples of this may include the modification of animal milk to enhance nutritional value, and the removal of specific proteins which may act as allergens, e.g. (β-lactoglobulin in cattle).

3. Xenotransplantation: the use of animal organs and other tissues for human transplantation. Physiologically, pig organs are similar to those of humans and are considered suitable for transplantation. However, there is a major problem with organ rejection. Although all of the mechanisms which are involved in this rejection are not completely understood, it is known that a major antigen involved is α-1,3,galactose. This is present on pig cells but is not found in humans who therefore mount an immune response. Nuclear transfer from cultured cells will facilitate knockout of the pig gene coding for α-1,3-galactosyl transferase. Such animals have now been reported by a number of groups including The University of Missouri (CNN) and PPL Therapeutics (CBC News). Potential organs and tissues for transplantation include the heart, lungs, kidneys, liver and pancreatic islets (treatment of diabetes) (for review White & Langford 1998).

4. Disease models: at present, many of the animal models available for the study of human genetic disorders are only available in mice. This species may not manifest the same clinical symptoms as in humans. Precise genetic modification of somatic cells coupled to nuclear transfer will allow the production of disease models in species which are physiologically more similar to the human in order to follow disease progression or to assess the benefits of any potential new therapies (including gene therapy). An example of this is cystic fibrosis – in mice this disease manifests in the gut and not the chest.

Cloning animals by nuclear transfer may also be of benefit in conservation and agriculture, for instance:

1. Preservation of genetic diversity and endangered species: although nuclear transfer from adult somatic cells has only been reported to date in a small number of species, the possibility exists that many other species may prove amenable to this technology. In terms of species preservation, however, a major limitation is the requirement of donor eggs from the same species by cryopreservation of cells from rare or endangered species. This may be of particular use in rare livestock species.

In addition, the combination of somatic cell cloning and genetic modification may facilitate modifications to animals which are beneficial to the agricultural community and indirectly to human health and prosperity, these may include:

1. Production of disease-resistant animals: this may be accomplished by either gene addition or removal (i.e. PrP gene involved in scrapie and BSE);

2. The modification of production traits (i.e. improving uniformity, or modification of animal products).

Role of nuclear transfer in stem cell therapies

The ability to produce animals by nuclear transfer demonstrates the ability to de-differentiate and subsequently re-differentiate the genetic material contained within a range of somatic cell types. The use of embryonic stem cells as a source for the production of specific differentiated cell lineages provides numerous opportunities for human therapies. At the present time stem cell populations have been isolated from embryonic fetal and adult tissues and studies are underway in numerous laboratories on the controlling lineage-specific differentiation. The practical aspects of cell transplantation with regard to function, longevity and rejection may differ dependent upon the cell origin. The establishment of cell banks of numerous tissue types may be required to reduce the problems that may be associated with cell rejection. Although stem cells derived from cord blood or adult tissues may provide a possible route for autologous transplantation, nuclear transfer followed by embryonic stem (ES) cell isolation may prove to be a valuable tool. The production of ES cells following dedifferentiation of somatic cells by nuclear transfer in the mouse has now been demonstrated (Munsie et al. 2000). The application of nuclear transfer in humans will provide valuable information on embryonic development and cell differentiation.