含dMAR真核表达载体的构建及影响EGFP表达的研究

首都医科大学学报 ›› 2005, Vol. 26 ›› Issue (2): 159-162.

含dMAR真核表达载体的构建及影响EGFP表达的研究

孙丽翠, 祁雅慧, 张静宜, 闫豫东, 殷红

首都医科大学实验中心

收稿日期:2004-06-21 修回日期:1900-01-01 出版日期:2005-04-24 发布日期:2005-04-24

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

Sun Licui, Qi Yahui, Zhang Jingyi, Yan Yudong, Yin Hong

Experiment Center, Capital University of Medical Sciences

Received:2004-06-21 Revised:1900-01-01 Online:2005-04-24 Published:2005-04-24

摘要/Abstract

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KpnI酶切回收的pEGFP-C1载体连接,HindⅢ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48hEGFP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。

关键词: EGFP, MAR, 基因表达调控

Abstract:

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.

Key words: EGFP, MAR, gene expression and regulation

中图分类号:

孙丽翠;祁雅慧;张静宜;闫豫东;殷红. 含dMAR真核表达载体的构建及影响EGFP表达的研究[J]. 首都医科大学学报, 2005, 26(2): 159-162.

Sun Licui;Qi Yahui;Zhang Jingyi;Yan Yudong;Yin Hong . Construction of Eukaryotic dMAR Expression Vector and its Retardance on EGFP Expression[J]. Journal of Capital Medical University, 2005, 26(2): 159-162.