Transfection Of Human Pre-insulin Gene Into Sheep Fibroblast Cells And Establishment Of The Transfected Cell Lines

Somatic cells nuclear transfer offers an efficient and practical method of producing transgenic animals, and the establishment of transgenic somatic cell lines are the basic of this method. Here we systematically study to establish sheep somatic cell lines in vitro, transfer human pre-insulin (hINS) gene into in vitro cultured cells and analyze karyotype, of which are three main steps to establish the transgenic cell lines. We have improved manipulation programs and parameters of those techniques, and established a fairly good system of foreign DNA transfection system for somatic cell lines and transfected cell clones.I. Established sheep somatic cell lines in vitroTissue from adult sheep ear skin were cultured and passaged using improved method of cutting into small pieces directly. We had a 100% of present for primary passage of fibroblast cells and successful passage rate for all the experiment adult sheep of different breed with this method (in other experiments, we also cultured ear tissue from other animals including sheep, goat, cow and north goat and we achieved satisfactory results). We had used 0.05% typsine-EDTA at different digestion times to purified fibroblast cells from epithelial cells according to their capacity of adhering to the plastic bottom. After several times of freeze-thaw, these purified fibroblasts have normal capacity of passage.II. Transfer human pre-insulin gene into sheep's fibroblast cells line and establish cell cloneBLG-hINS (β-lactoglobulin/human pre-insulin) gene was co-transfected with GFP (green fluorescent protein) gene as the selective marker using liposome to deliver DNA into target cells. After three weeks selecting by G-418, the stable transfected cells were obtained. To determine the successful transfection with these two foreign genes, DNA sample of these cells was screened by PCR. These cells were subsequently cloned by following two ways: one is to passage the cells into 35mm tissue culture dishes with very low cell density (about 50~100cells/ml), and picked the individual healthy colonies into an other tissue culture dish after about a week culture; two is to pick single cell into 96-well tissue culture dishes under microscope, allow them to expand into colonies. Healthy colonies were determined to assure the presnce of both BLG-hINS and GFP genes by PCR.III. Karyotyping analysisHigh passaged cells (adult sheep cells cultured up to 30 passages and sheep fetus cells cultured up to 45 passages), transfected cells and cloned cells have not been observed abnormal of chromosomes' number and ploidy by karyotyping analysis.