小鼠胚胎干細(xì)胞轉(zhuǎn)染條件的優(yōu)化

對(duì)小鼠胚胎干細(xì)胞（mESCs）轉(zhuǎn)染條件進(jìn)行優(yōu)化
分別將0.5×105,1×105,2×105,4×105個(gè)細(xì)胞接種24孔板（各細(xì)胞量均接種2孔）,培養(yǎng)48h在其細(xì)胞融合度分別為30%,50%,70%,90%左右時(shí),分別設(shè)置相同的（0.8μgDNA/2μL脂質(zhì)體）和不同的DNA/脂質(zhì)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(zhì)體（LipofectamineTM2000）轉(zhuǎn)染法,將pEGFP-N1報(bào)告基因載體轉(zhuǎn)染到mESCs中,48h后通過(guò)熒光觀察和流式分析檢測(cè)各組EGFP熒光強(qiáng)度,比較轉(zhuǎn)染效率。
結(jié)果在質(zhì)粒及脂質(zhì)體用量相同（0.8μgDNA/2.0μL脂質(zhì)體）的條件下,隨著細(xì)胞融合度的增加,EGFP熒光強(qiáng)度逐漸減弱,在細(xì)胞融合度為30%,50%,70%,90%左右時(shí)轉(zhuǎn)染,各組EGFP陽(yáng)性細(xì)胞率分別為56.4%,51.8%,40.8%和23.3%。提高轉(zhuǎn)染質(zhì)粒及脂質(zhì)體量可在一定程度上提高轉(zhuǎn)染效率,但脂質(zhì)體的細(xì)胞毒性也隨之增加,不利于細(xì)胞生長(zhǎng)。
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