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

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

NUCLEAR TRANSFER TECHNOLOGY

Participant:

Roger A. Pedersen, Ph.D.
University of California, San Francisco

A. What are the new insights from successful nuclear transfer cloning of Dolly and her successors?

1. The production of normal adult animals by nuclear transfer of adult somatic cells provides new insights about totipotency of (some) adult nuclei. Previous information about animal development provided no basis for the expectation that adult nuclei (other than germ cell nuclei) could support development to adulthood.

2. Additional insight is also gained about the biological issues of differentiation and development. Because adult nuclei can be induced to recapitulate development by transfer to oocyte cytoplasm, the factors responsible for their stable differentiation in the original tissue environment are apparently subject to alteration.

3. The recent studies (1996-98) reinforce previous information about the inefficiency of somatic nuclei at recapitulating development (1-2% development to term by somatic cell nuclei versus 50-75% for normally fertilized oocytes). In light of this inefficiency, and related high rates of embryonic wastage, fetal and neonatal deaths and birth defects, the technology is clearly not applicable to reproductive cloning of humans.

4. Nuclear transfer provides new insights into normal development of fertilized embryos: eggs and sperm are not unique in possessing nuclear totipotency, but are unique in their capacity to activate eggs (in the case of sperm) or to evoke nuclear potential for embryogenesis (in the case of oocytes).

1. Genetic reprogramming can be defined as transformation from the pattern of gene expression that is characteristic of the donor cell to one that is appropriate for early embryonic development. Genetic reprogramming of germ cell nuclei is presumed to occur normally during gametogenesis, as indicated by re-activation of inactive X chromosome in germ cells, erasure of genomic imprinting and restoration of totipotency. Oocytes have the ability to restore/evoke totipotency of somatic cell nuclei, thus resembling earlier stages of germ cells in having this capacity. Does genetic reprogramming involve active silencing of previously-expressed genes and activation of embryo-specific genes? Can the efficiency of development following nuclear transfer be improved through alterations in genetic reprogramming?

2. How widespread or restricted is the nuclear capacity to recapitulate development when transferred to oocyte cytoplasm? Mammary gland nuclei (in vitro) and cumulus mass nuclei (ex vivo) support full term development, but Sertoli nuclei and neuronal nuclei do not, at least in mice. What is the response of nuclei from other tissue sources to an oocyte cytoplasmic environment? What do nuclear donor sources that support/do not support embryonic development have in common?

3. Can somatic cell nuclei be transformed efficiently enough to use them as the vectors for precise genetic manipulation? For this purpose, it will be necessary to maintain euploid somatic cells in prolonged culture and to achieve homologous recombination in them. To date, only a few studies have indicated that cultured somatic cells can sustain homologous recombination. Do somatic cells have the biochemical status and chromatin structure to undergo homologous recombination? Another prerequisite for the use of somatic cells as donor material is maintenance of proliferative potential in euploid, non-transformed cells.