EcoRⅡ甲基化酶基因在小鼠NIH3T3细胞中的表达

Expression of EcoRⅡ Methylase Gene in Mouse NIH3T3 Cells

CG外甲基化在哺乳动物细胞中的生物学意义仍不清楚。建立了特定位点CCWGG（W＝A／T）甲基化的哺乳动物细胞株。来自大肠杆菌的EcoRⅡ甲基化酶（催化CCWGG→CmCWGG）基因经定点诱变后重组到真核表达载体pCI－neo上，采用脂质体介导的方法将其转染小鼠NIH3T3细胞，经G418筛选获得有效表达的细胞株，酶切证实该细胞株基因组DNA上的大部分EcoRⅡ识别位点已被甲基化，PCR鉴定EcoRⅡ甲基化酶基因已稳定整合到细胞染色体上。

The biological significence of non-CG Methylation in mammalian cells is unknown. Cell linein which specific site CCWGG(W=A/T) had been methylated was established. E. colt EcoR Ⅱmethylase (catalyze CCWGG→Cm CWGG) gene was recombined into eucaryotic expression vectorPCI-neo after site-directed mutagenesis, and with it mouse NIH3T3 cells were trans feeted byliposome mediation. Effectively expressing cell line was obtained through G418 selection. MostEcoR Ⅱ sites in genomic DNA of this cell line had been specifically methylated. Through PCR, itwas confirmed that EcoR Ⅱ methylase gene had been integrated into chromasomal DNA steadily.