小鼠pαMHC-EGFP胚胎干细胞株的构建

Generation of pαMHC-EGFP Murine Embryonic Stem Clones

应用电穿孔方法将含有α肌球蛋白重链启动子的pαMHC-EGFP载体转染到D3系小鼠胚胎干细胞,应用200μg/ml新霉素进行药物选择。采用悬浮培养法,体外诱导分化心肌细胞。荧光显微镜下,观察到第7天和第8天拟胚体中出现“跳动”的心肌细胞并同时有绿色荧光蛋白的表达。同时与D3系小鼠胚胎干细胞比较心肌细胞分化率的变化无显著差异(P>0.05)。该细胞株在分化心肌细胞的同时,具有绿色荧光蛋白的标记,因而利于对心肌细胞的识别和纯化。

We have established a murine D3 embryonic stem cell clone（αMHC-EGFP） with expresses the enhanced green fluorescent protein （EGFP） under the transcriptional control of the alpha-myosin heavy chain （αMHC） promoter, selected ES clones with 200 μg/ml neomycin, cardiomycytes derived from transgenic pαMHC-EGFP embryonic stem （ES） cells with “hanging drop” method in vitro. We have confirmed pαMHC-EGFP ES clones by expression of green fluorescence protein （GFP） in seventh day or eighth day under fluorescence microscope. Meantime compared with D3 ES cells, the change of ratio of cardiomycytes differentiation is not remarkable （P〉 0.05）. So the pαEGFP murine embryonic stem clone is of great benefit to identify and purify of cardiomyocytes from undifferentiated ES cells.