Cultivation with Untransfected Fibroblasts Stimulates Proliferation of a Single Gene-Modified Fibroblast Derived from a Clawn Miniature Swine Foetus

Porcine embryonic fibroblasts (PEFs) have been used extensively as donor nuclei for the production of cloned pigs via somatic cell nuclear transfer (SCNT). Somatic cell nuclear transfer of gene-targeted PEFs has been the only way to produce gene-targeted pigs, given the lack of germ-line-competent porcine embryonic stem cells. Unlike other primary-cultured cells, such as murine embryonic fibroblasts, a single porcine PEF is unable to proliferate under normal conditions in which a certain number of PEFs (likely over 100) can grow normally. This limitation greatly hampers re-cloning of gene-modified PEFs, which is required for SCNT. Herein, we demonstrate the cultivation of a single PEF transfectant carrying the pEGFP-N1 plasmid with intact normal PEFs (>100) in a Terasaki microtest plate, which resulted in stimulation of the growth of the former cell (doubling time = 2.6 days). In contrast, when a single cell was cultured, it could typically divide only once and never divided more than twice. When a single transfectant was seeded in a well of a 96-well plate together with 5 × 10(4) untransfected PEFs and was subsequently selected in the presence of G418, we obtained a pure cell population of single-cell origin. Thus, this method should be useful for the purification of target recombinant pig clones from mosaic populations that cannot be cultured as a single cell or for which suitable cell growth-promoting conditions are unclear.