The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

Human Prourokinase (proUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase that is associated with clinical benefits in patients with acute peripheral arterial occlusion. For production of transgenic cow as human proUK bioreacotor, we conducted this study to establish efficient production system for bovine transgenic embryos by somatic cell nuclear transfer (NT) using human prourokinase gene transfected donor cell. An expression plasmid for human prourokinase was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human prourokinase target gene into a pcDNA3 plasmid. Cumulus cells were used as donor cell and transfected with the expression plasmid using the Fugene 6 as a carrier. To increase the efficiency for the production of transgenic NT, development rates were compared between non-transfected and transfected cell in experiment 1, and in experiment 2, development rates were compared according to level of GFP expression in donor cells. In experiment 1, development rates of non-transgenic NT embryos were significantly higher than transgenic NT embryos (43.3 vs. 28.4％). In experiment 2, there were no significant differences in fusion rates (85.4 vs. 78.9％) and cleavage rates (78.7 vs. 84.4％) between low and high expressed cells. However, development rates to blastocyst were higher in low expressed cells (17.0 vs. 33.3％), and GFP expression rates in blastocyst were higher in high expressed cells (75.0 vs. 43.3％), significantly.