小鼠胚胎干細胞轉染條件的優化

對小鼠胚胎干細胞（mESCs）轉染條件進行優化
分別將0.5×105,1×105,2×105,4×105個細胞接種24孔板（各細胞量均接種2孔）,培養48h在其細胞融合度分別為30%,50%,70%,90%左右時,分別設置相同的（0.8μgDNA/2μL脂質體）和不同的DNA/脂質體用量（DNA量（μg）/Lipo量（μL）分別為0.4/1,0.8/2,1.6/4,3.2/8）,采用脂質體（LipofectamineTM2000）轉染法,將pEGFP-N1報告基因載體轉染到mESCs中,48h后通過熒光觀察和流式分析檢測各組EGFP熒光強度,比較轉染效率。
結果在質粒及脂質體用量相同（0.8μgDNA/2.0μL脂質體）的條件下,隨著細胞融合度的增加,EGFP熒光強度逐漸減弱,在細胞融合度為30%,50%,70%,90%左右時轉染,各組EGFP陽性細胞率分別為56.4%,51.8%,40.8%和23.3%。提高轉染質粒及脂質體量可在一定程度上提高轉染效率,但脂質體的細胞毒性也隨之增加,不利于細胞生長。
低融合度（30%～50%）條件下,使用0.8μgDNA/2
 
The study was done to optimize the transfection efficiency of mouse embryonic stem cells（mESCs）.[Method] mESCs were seeded into 24-well plates at a density of 0.5×105,1×105,2×105,4×105 per well（each for two wells）.48 h later,cells were transfected when cell confluence was 30%,50%,70%,90% respectively.The reporter plasmid pEGFP-N1 was transfected into cells using LipofectamineTM 2000 by using the same amount（0.8 μg DNA/2 μL liposome） or different amounts of DNA and liposome（DNA（μg）/li