From Cell to Production

Technical Challenges of Cloning Pigs for BioMedical Research

Somatic Cell Nuclear Transfer in Mammals

SATACs and Transgenesis

Concerns About Gene Transfer and Nuclear Transfer in Domestic Animals

Prospects and Hurdles in Optimizing the Vascular Support of Engineered Tissues

ES Cells Make Neurons in a Dish

Nuclear Transfer and Gene Targeting in Domestic Animals: Bioreactors of the Future

Genomics: Delivering Cell Culture Systems for Tissue Therapy

Nuclear Transfer Technology

Gene Targeting in Domestic Species: Challenges and Opportunities

Homologous Recombination and Genetic Engineering of Transgenic Recombinant Animals

Nuclear Transplantation in the Cow: Future Challenges

Enhancing Transgenics through Cloning

ES Cells Offer is a Power Tool for Understanding the Genetic Control of Tissue Development and for Screening Potential Therapeutic Drugs

Human Germline Engineering -- The Prospects for Commercial Development

Mammalian Artificial Chromosomes for Animal Transgenesis

Understanding Developmental Abnormalities in Offspring Produced by Nuclear Transplantation

Role of Cell Cycle

Cloning and Other Reproductive Technologies for Application in Transgenics

APPLICATION OF NUCLEAR TRANSFER TECHNOLOGY IN THE GENERATION OF DONOR PIGS FOR XENETRANSPLANTATION

Participants:

Robert J. Hawley and Julia L. Greenstein
BioTransplant, Inc.
Building 75, 3^rd Avenue
Charlestown Navy Yard
Charlestown, MA 02129

The critical shortage of human organs available for transplantation in the treatment of end-stage organ disease has greatly accelerated research and development in the field of xenotransplantation. A combination of physiological, biological, economic and ethical considerations make the pig the most likely animal for sustainable use as a xenogeneic organ donor (1).

Suppression of cell-mediated rejection mechanisms is currently accomplished in human allograft transplantation with drug therapy. Transplantation of pig organs into humans requires elimination or suppression of hyperacute and acute vascular rejection mechanisms particular to xenografts, as well as means to modulate a more vigorous cellular response (2). Modifications of the pig genome, through functional deletion of pig genes and introduction of human genes, have been proposed to deal with the various immunological barriers. Dominant modification of the genome has already been undertaken, with some promising results in terms of extended organ survival in primate animal models (3).

The only technology currently available for transgenic modification of swine is pronuclear microinjection. While useful dominant modifications can certainly be made in this manner, the process is relatively inefficient and does not permit site directed genetic modifications to be made. Efforts to develop ES (and EG) cell lines in pigs (and other species) are ongoing, but have not to date yielded lines capable of germ line transmission. Implementation of nuclear transfer technology in pigs would have a significant impact on development of xenotransplantation organs by permitting complex modifications, such as knockouts or targeted expression modifications, to be made in pigs. Additionally, it would allow small numbers of karyoplast donor animals to be maintained, while utilizing alternative cytoplast donors and recipients to meet the bulk of animal use requirements. This should greatly increase rate at which modifications can be introduced into limited stocks of genetically valuable animals and permit much faster introduction of serial modifications.

While successful nuclear transfer in pigs using early blastomere nuclei has been reported (4), a number of challenges need to met before efficient generation of animals from modifiable somatic cells can be expected. Methods for in vitro maturation of pig oocytes, allowing for economical acquisition of large numbers of recipient cytoplasts, have been developed (5) but are not fully characterized with respect to use in generating live animals. Efficient activation of porcine oocytes has required substantial innovation (6), but the potential for these methods to support development to term is unknown. There is also considerable room for improvement in the area of in vitro culture techniques for porcine embryos. Efficient and consistent culture to later blastocyst stage would not only aid in selection of embryos for transfer to recipient animals, but also greatly assist in further development of successful nuclear transfer protocols. Finally, the earlier onset of embryonic transcription in pigs (7) may require modifications to nuclear transfer or karyoplast preparation protocols to enable faster reprogramming of the donor nucleus.

Development of the pig as a xenograft donor will be greatly accelerated by development of successful nuclear transfer procedures using modifiable somatic cells. Although there challenges specific to success in pigs, the rapid pace of progress in other species suggests that these challenges can be met in the near future.

1. Cooper D.K.C., Y. Ye, L.L. Rolf Jr. and N. Zuhdi. "The Pig as Potential Organ Donor for Man". In Xenotransplantation, D.K.C. Cooper et al. eds., Springer-Verlag, New York, 1991.