Development of efficient strategies for the production of genetically modified pigs

Although pronuclear DNA micro-injection has long been the most reliable method to produce transgenic pigs, the efficiency of production of transgenic offspring is generally plagued by 1% of the DNA-injected embryos. Therefore, a problem with this method is the need for large numbers of pronuclear stage embryos. One great advancement would be the use of in vitro-matured (IVM) oocytes for the purpose of transgenic pig production. High developmental competence of IVM oocytes was proven by transfer of parthenogenetic IVM oocytes. A combined method of sperm vectors with the IVM of oocytes would make the production of transgenic pigs remarkably feasible. Rate of blastocyst formation following intracytoplasmic sperm injection (ICSI) by frozen sperm was over 20%, and transgene was expressed in approximately 50% of blastocysts generated. Somatic cell nuclear transfer would enable more efficient and sophisticated genetic modification of the pig. Simultaneous comparison between two nuclear transfer methods by electro-fusion and intracytoplasmic injection revealed clear differences in the pattern of nuclear remodeling and development of the reconstructed embryos. To specify the donor cell type that allows efficient genetic modification and easy reprogramming or to establish such cell lines is a critical issue in pig cloning. We tested pre-adipocytes from the subcutaneous adipose tissue of adult pigs for nuclear transfer. Cell cycle synchronization by differentiation induction is unique to the pre-adipocytes. Frequency of apoptosis was low in the cells synchronized by differentiation induction compared with other synchronization methods, including serum starvation, confluency, and chemical treatment. It would be of great worth if cryopreserved clone embryos were available. We have demonstrated that cryopreservation of in vitro-produced porcine embryos as well as clone blastocysts is possible by our unique method.