小鼠胚胎干细胞转染条件的优化

Optimization of the transfection efficiency of mouse embryonic stem cells

【目的】对小鼠胚胎干细胞(mESCs)转染条件进行优化。【方法】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。【结论】低融合度(30%～50%)条件下,使用0.8μgDNA/2.0μL脂质体对mESCs进行转染,可获得较高的转染效率(>50%),同时可维持良好的细胞状态。

[Objective] 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 0.5×10^5,1×10^5,2×10^5,4×10^5 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 Lipofectamine^TM 2000 by using the same amount（0.8 μg DNA/2 μL liposome） or different amounts of DNA and liposome（DNA（μg）/liposome（μL）：0.4/1,0.8/2,1.6/4,3.2/8） respectively.48 hr later,the fluorescence intensities in each group were observed and the transfection efficiencies were compared.[Result] The result of fluorescence microscopy and FACS analysis showed that the percentage of EGFP positive cells gradually decreased（56.4%,51.8%,40.8% and 23.3% respectively） as the cell confluence increased（30%,50%,70% and 90% respectively） when using the same amount of DNA and liposome（0.8 μg DNA/2.0 μL liposome/well of 24-well plate）.When using more DNA and liposome,transfection efficiency further increased.However,obvious cytotoxicity could be observed.[Conclusion] High transfection efficiency（50%） on mESCs could be achieved by using 0.8 μg DNA/2.0 μL liposome/well of 24-well plate under condition of low cell confluences（30%-50%）.