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

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

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

NUCLEAR TRANSPLANTATION IN THE COW: FUTURE CHALLENGES

Participant:

James M. Robl
Department of Veterinary and Animal Sciences
Paige Laboratory
University of Massachusetts
Amherst, MA 01003

Recent successes with somatic cell nuclear transplantation open the door to a vast array of important potential applications in agriculture and medicine. In agriculture, particularly in the cattle industry, there is a need for the improvement of beef and milk production efficiency, increased uniformity of beef products and the development of specialized beef and milk products. The U.S. beef cattle industry consists of about 100 million animals of which about 40 million are slaughtered each year for a value of approximately $35 billion. According to the National Cattleman’s Association National Beef Quality Audit Summary "genetic-related non-conformities or quality defects account for $248.32, or almost 88% of the industry’s total economic loss per animal". Total industry loss is calculated to be nearly $10 billion. Many of these losses could be reduced by increased genetic uniformity and improved genetic quality. The U.S. dairy industry consists of about 8 million animals and about $15 billion product value each year. Improved genetics has resulted in a doubling of milk production efficiency over the past 30 years and can continue to improve efficiency in the future. Altered genetics may be useful for the production of novel milk-derived products in the future.

For widespread use of cloning in agriculture, improvements in efficiency need to be made. In our work with the cow, the efficiency of blastocyst production for several lines of donor fibroblasts, both from fetal and adult animals, is about 8 to 10% with in vitro matured oocytes shipped to the laboratory by express mail. This compares to about 25% development of parthenogenetically activated ooctyes from the same source. Presumably, 75% of the embryos fail to develop because of poor egg quality. The other 15% loss is due either to problems with manipulation or nuclear/cytoplasmic incompatibility. Our preliminary work indicates that manipulation is only a small part of this loss and most of this is due to problems with fusing small cells to the oocyte cytoplast. Therefore, work needs to be done to reduce nuclear/cytoplasmic incompatibility by determining the mechanisms of nuclear reprogramming and improving the process.

Production of a blastocyst is not an accurate endpoint to judge the success of a manipulation procedure. In our work, cells from various sources will support development into morphologically normal blastocysts but the quality of these blastocysts varies greatly as assessed by further development in vivo. Because blastocyst morphology is not a good indicator of manipulation success and development to term is an impractical endpoint better methods need to be developed to assess the quality of manipulated embryos, particularly methods that reflect in vivo development success.

With the limited information that we have accumulated, it appears that most losses after transfer occur before 60 days of gestation and after 8 months of gestation. Nuclear/cytoplasmic incompatibility and the long term effect of the in vitro environment likely influence development to term. Again, the mechanisms of nuclear reprogramming are poorly understood and need considerable investment in effort in the future. Furthermore, although there have been successes in the development of in vitro systems for maturation of oocytes and culture of embryos further work needs to be done with endpoints that are more representative of development to term.

Improvements in agricultural production efficiency could be made through the genetic modification of cattle. Nuclear transfer technology has already been used to produce cattle with a randomly inserted gene. Further work needs to be dome on methods of inserting genes into specific locations. Targeted insertions are made by homologous recombination of the introduce genetic sequence with an endogenous sequence. Typically this approach requires a first selection of cells with the genetic sequence integrated and a second selection for cells with the genetics sequence integrated into the correct location. This process requires a significant number of population doublings in culture. Therefore, efforts need to be made to either extend the life span of primary cells in culture or to streamline the selection procedure.