From Cell to Production

Technical Challenges of Cloning Pigs for BioMedical Research

Somatic Cell Nuclear Transfer in Mammals

SATACs and Transgenesis

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

CONCERNS ABOUT GENE TRANSFER AND NUCLEAR TRANSFER IN DOMESTIC ANIMALS

Participant:

Neal First
University of Wisconsin at Madison

1. Cell-cycle synchrony was not necessary in the older cloning methods (Prather et al. 1987, Stice et al. 1996- cattle, Willadsen, 1986-sheep) because an aged enucleated oocyte was used. This oocyte does not allow nuclear envelope breakdown or a new zygotic one-cell DNA replication S phase of the cell cycle. Rather, the oocyte cleaves to two cell with mitotic division of the introduced nucleus. Enucleated early nonaged oocytes are essential to allow maximum genomic and cellular reprogramming of differentiated nuclei. An S phase and DNA replication will occur. Mistakes in incomplete DNA replication at the end of meiosis will either end in failed later embryo development or in interphase check point screening as the new cell enter mitosis. In mice, the cell fusion studies of Fulka et al. (published 1996, 1997, 1998 in Bio Essays and Human Reproduction and recently submitted to Human Reproduction) show that the mouse oocyte during meiosis has no cell cycle checkpoints, but screening for normal DNA replication does occur at interphase and later at M phase of the mitotic cell cycle.

   So the need for cells of the G₀ to G ₁ stage to allow complete DNA replication depends on the developmental condition of the donor cell and the kind of nuclear transfer being done.

   G₀ to G ₁ synchrony of donor cells is also needed to prevent the contribution of a centrosomal organizing center by the donor cell. This results in two centrosomal organizing centers and ploidy problems for the resulting embryo (Navara et al., 1994, Development). Whether near-death differentiated cells such as cumulus and 5-day-starved cells are prepared for demethylation and acetylation changes by their apoptotic condition or by the G₀–G₁ state of the cell is unknown. Certainly the G₀–G₁ state is essential to get complete DNA replication at the first mitotic cycle.

2. It remains to be determined yet whether there can be incompatibilities between the cytoplasm of a donor cell and the oocyte, for example, mitochondrial difference. Certainly the use of nearly pure nuclear karyoplasts as reported for mice by Dr. Yanagimachi's lab in Hawaii will help answer questions in this area. These answers are especially important in the development of systems for intraspecies nuclear transfer as described by Dominko et al. Theriogenology, Jan. 1998.

3. The efficiencies of all methods of nuclear transfer are still very low being at best 1:10 for embryonic cells and 1:50 for either differentiated fetal cells (Cibelli et al. 1998, Science) and 1:50 for adult cumulus cells as nuclear donors (Wakayama et al. 1998, Science 394: 369-374). Losses appear to occur throughout development, and failures appear to be due to mistakes in gene expression as well as in cellular reprogramming.

4. The papers of Cibelli et al. in Science, 1998 and Nature Biotechnology, 1998 show potential utility for gene transfer or deletion in cultured fetal fibroblast cells used in nuclear transfer in cattle. The described system also allows another round of gene transfer into the resulting cultured embryonic stem cells which can be then used to make offspring.

5. To date, the most efficient systems for making transgenic animals are the pseudotype viral vector system described by Anthony Chan (University of Wisconsin Ph.D. Thesis, 1997) and perhaps a sperm-mediated DNA transfer system claimed by Shemesh at Kimron Veterinary School Bet Degan Israel to produce first and second generation transgenic chickens.

   The viral vector system of Chan 1997 is approximately 20% efficient. The success rests with use of a highly infectious viral vector yielding titers of +10⁹–10¹⁰ and the introduction of DNA into the M II oocyte rather than a zygote nucleus. Unconfirmed reports from the Check Republic also claim transgenic offspring in swine from sperm-mediated DNA transfer. If true the value of sperm-mediated DNA transfer is that it can be used either with artificial insemination or in vitro fertilization. In my view, the ability to commercially make transgenic animals for agricultural and pharmaceutical milk products uses is presently here and becoming much more efficient. There is an issue of chimeric expression of DNA in embryonic cells and its impact on failed expression in the derved tissue or germ line.