Production of cloned pigs by using somatic cells as donors.

Abstract

NUCLEAR TRANSFER is a procedure by which genetically identical individuals can be created. The applications of these techniques for nuclear transfer will be in agricultural, biomedical and basic research. Based on the sources of donor cells, nuclear transfer can be classified into embryonic cell nuclear transfer and somatic cell nuclear transfer. Offspring from cultured cells were first reported in 1994 (Sims and First, 1994) in cattle. Since this paper showed that cells could be cultured at least 28 days prior to nuclear transfer, it held great promise for genetically modifying the cells prior to nuclear transfer, and thus the possibility of producing animals with specific genetic modifications. This concept is exemplified by the generation of transgenic sheep (Schnieke et al., 1997), pigs (Park et al., 2001), calves (Cibelli et al., 1998a), and gene-targeted sheep (McCreath et al., 2000) and pigs (Lai et al., 2002b), derived from nuclear transfer approaches by using transfected somatic cells. For pigs, somatic cell nuclear transfer has another specific significance, as is it would allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation (Lai et al., 2002b). However, the efficiency of somatic cell nuclear transfer, when measured as development to term as a proportion of oocytes used, has been very low (1–2%). A number of variables influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, the method of artificial oocyte activation, embryo culture, possible loss of somatic imprinting in the nuclei of reconstructed embryos, failure of adequate reprogramming of the transplanted nucleus, and the techniques employed for nuclear transfer. In pig, there are additional difficulties in that the quality of embryos produced in vitro is low, and at least four good embryos are required to initiate and establish a pregnancy. Procedures for nuclear transfer include the following steps: acquisition of recipient oocytes and donor cells, enucleation (removal of the chromosome from recipient oocytes), insertion of donor nuclei into enucleated oocytes, artificial activation of reconstructed oocytes, and embryo transfer (transfer of the reconstructed embryos into a surrogate).