摘要
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。
Objective To study the regulation role of dMAR on EGFP expression. 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 EGFP expression 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 EGFP was significantly inhibited by dMAR(-) co-expression. In contrast, the expression of EGFP was not affected by dMAR(+) co-expression.
出处
《首都医科大学学报》
CAS
2005年第2期159-162,共4页
Journal of Capital Medical University
基金
首都医科大学青年基金(No.0213)资助项目
