Effects of Gene Methylation Reprogramming in Cloned Calves Derived from In Vitro-Transfected Somatic Cells

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP) reporter transgene and used as nuclear donor cells in oocyte reconstructions. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either metaphase or telophase activation, and PCR technology was also employed. The results showed that GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. Embryonic development to the blastocyst stage was not significantly different among metaphase and telophase groups. Therefore, GFP transgene technology can be used to select embryoes derived from transgenic animals.