含dMAR真核表达载体的构建及影响EGFP表达的研究

目的　研究dMAR对EGFP基因表达的调控作用。方法　用Kpn -I酶切pGFP/MAR,回收含MAR下游850bp的片段dMAR,与Kpn I酶切回收的pEGFP-C1 载体连接,Hind Ⅲ酶切鉴定方向,构建正向、反向连接的真核表达载体pEGFP/dMAR′和pEGFP/dMAR。将该表达载体转染COS7细胞,荧光显微镜观察EGFP的表达并采用流式细胞术进行荧光定量。结果　pEGFP- C1,pEGFP/dMAR′和pEGFP/dMAR转染后48 h EGFP的表达量分别为45.1%、40.5%和11.3%。结论　pEGFP/dMAR显著抑制了EGFP的表达,pEGFP/dMAR′的增强表达作用不明显。

Construction of Eukaryotic dMAR Expression Vector and its Retardance on EGFP Expression

Objective To study the regulation role of dMAR on EGFPexpression. Methods The dMAR eukaryotic expression vectors were constructed by inserting 850 bp dMAR fragment into pEGFP-C1 vector at the downstream of EGFP with two different orientations. pEGFP/dMAR(+)(sense) and pEGFP/dMAR(-)(antisense) were made. The regulation of EGFP by overexpression dMAR was assessed with fluorescent microscope and flow cytometry(FCM) after the pEGFP/dMAR(+) or pEGFP/dMAR (-) vector transfected into COS7 cells. Results The EGFPexpression of pEGFP-C1, pEGFP/dMAR(+) and pEGFP/dMAR(-) was 45.1%、40.5% and 11.3% respectively 48 h post-transfection. Conclusion The expression of EGFPwas significantly inhibited by dMAR(-) co-expression. In contrast, the expression of EGFP was not affected by dMAR(+) co-expression.