| Research Abstract |
In order to develop the gene targeting technique in fish, we have established cell lines derived from blastura embryos of a small labortory fish, medaka (Orzias latipes). We planed this study to investigate experimental conditions for gene transfer into these cell lines by homologous recombination.
1) Conditions for gene transfer using electroporation : We examined the efficiency of gene transfer using a gene construct of medaka beta-actin promoter fused to GFP in various voltages from 0 to 450V.The rate of survived cells and gene transfer became lesser and greater with increase of the voltage, respectively. At the maximum voltage, 450v, the rate of survived cells and gene transfer were 12.8% and 0.77%, respectively.
2) Conditions for gene transfer using lipfection : In the treatment of cells for 20 hours with the medium containing 40mu1 lipofectamine and 18mugDNA/ml, 0.5% of cells cxpressed GFP.These GHP-expressing cells were isolated and cloned.
3) We examined the sensitivity of cells to G418 and GANC for selection of cells with homologous recombination. Cells were sensitive to G418 at concentrations higher than 300mug/ml when treated with various concentrations of 0 to 600mug/ml. At the concentration of 600mug/ml, all cells died within 5 days. Cells transfected wity DNA of the neomycin-resistant gene were resistant to G418 of 600mug/ml. We exmined cytotoxicity at concentrations from 0 to 0.5muM.The cytotoxicity was observed at concentrations higher than 0.2muM.
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