title: Cultivation with untransfected fibroblasts stimulates proliferation of a single gene-modified fibroblast derived from a clawn miniature swine foetus.
abstract: 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.
authors: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan. masasato@ms.kagoshima-u.ac.jp
authors: M
authors: M
authors: Sato
authors: M
authors: M
authors: Yoshida
authors: K
authors: K
authors: Miyoshi
authors: M
authors: M
authors: Ohtsuka
authors: S
authors: S
authors: Watanabe
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013556:Swine, Miniature / Q000196:embryology*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins