小鼠胚胎干细胞转染条件的优化

小鼠胚胎干细胞转染条件的优化

对小鼠胚胎干细胞（mESCs）转染条件进行优化
分别将0.5×105,1×105,2×105,4×105个细胞接种24孔板（各细胞量均接种2孔）,培养48h在其细胞融合度分别为30%,50%,70%,90%左右时,分别设置相同的（0.8μgDNA/2μL脂质体）和不同的DNA/脂质体用量（DNA量（μg）/Lipo量（μL）分别为0.4/1,0.8/2,1.6/4,3.2/8）,采用脂质体（LipofectamineTM2000）转染法,将pEGFP-N1报告基因载体转染到mESCs中,48h后通过荧光观察和流式分析检测各组EGFP荧光强度,比较转染效率。
结果在质粒及脂质体用量相同（0.8μgDNA/2.0μL脂质体）的条件下,随着细胞融合度的增加,EGFP荧光强度逐渐减弱,在细胞融合度为30%,50%,70%,90%左右时转染,各组EGFP阳性细胞率分别为56.4%,51.8%,40.8%和23.3%。提高转染质粒及脂质体量可在一定程度上提高转染效率,但脂质体的细胞毒性也随之增加,不利于细胞生长。
低融合度（30%～50%）条件下,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