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

Application of Nuclear Transfer Technology in the Generation of Pigs for Xenetransplantation

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

Cell Culturing Technology as a Major Hurdle in the Commercialization of Genetically Altered Animals

FROM CELL TO PRODUCTION

Participants:

Mike Bishop, Nick Strelchenko, Erik Forsberg, Phil Damiani, Ken Eilertsen, Marv Pace, Paul Goluecke, Gail Jurgella, Jeff Betthauser, Martha Phfister-Genskow, Lynnette Brennan, Infigen, Inc. and Otto Postma, Pharming Health Care

Cloned animals from non-embryo derived cells are here. However, several steps in the cloning process are inconsistent and make successful repetition of animals from the same cell lines challenging. Process inefficiencies begin with the cell culture itself. There are clearly differences in culture systems, media and sera used to establish cells correctly for repeatable successful NT. In reality, there is continued debate regarding identification of the correct cell type and how the correct cell type is established. On a larger scale, further molecular definition of what constitutes a fibroblast cell, a primordial germ cell, an embryonic germ cell or a cumulus cell is still required. Molecular explanations that attribute to a cell's totipotency are highly desirable. In addition, the ability to diagnose an individual cell's presence in either G_o or G₁ stage of the cell cycle at the time of NT is also an unknown. In order to bring this technology to the commercial marketplace with the required repeatability and consistency for production these questions must be answered. Cell line variation for pregnancy initiation (%) and pregnancy to term (%) is substantial in all published (and non-published) studies. Once you select the correct cell type and use it for NT additional questions arise. How does that cell's nuclear material interact with the cytoplasm of the enucleated oocyte? What programming events are required to correctly initiate and proceed through subsequent developmental processes? Is this important? Is it a function of choosing the correct cell type in the beginning? What are the effects of activation procedures on totipotency? Also, what are the downstream variables that affect its ability to initiate 'normal' pregnancies? Many cell types can be used in the NT process to derive a somatic cell embryo. While the resulting embryos initiate pregnancies, a large percentage of them are aborted very soon afterwards. Finally, what are the effects on totipotency of cells subjected to the process of transfection and genome manipulation (knockouts, knock-ins, etc)? It appears that transfection of the totipotent cell is possible. Can a totipotent cell withstand multiple rounds of manipulation required for production of modified animals for production agriculture, the pharmaceutical or xenotransplantation fields? Further yet, can the cell still remain stable in culture? There may be practical limitations to how much 'stress" totipotent cells can withstand from a culturing, transfection, manipulation and cryopreservation standpoint requiring derivation of processes and procedures for 'rejuvenating' cells post-stress.

In summary, the use of NT cloning procedures with somatic cells is an exciting emerging technology that has numerous applications. In a very highly controlled system many of the variables and questions posed above are manageable and success is achievable. Joint relationships that build on synergies for researching, developing and applying the cloning technology to the agricultural, pharmaceutical, xenotransplant fields and others will provide a successful dynamic model for advancing the technology.