上海交通大学学报

上海交通大学学报（自然版） ›› 2012, Vol. 46 ›› Issue (05): 803-807.

• 生物科学 • 上一篇    下一篇

靶向srGAP家族的串联短发夹RNA表达

马跃，戴云凯，陈铿，米亚静，金卫林   

  1. （上海交通大学 生命科学与技术学院， 上海 200240）
  • 收稿日期:2011-03-20 出版日期:2012-05-28 发布日期:2012-05-28
  • 基金资助:

    国家自然科学基金资助项目(30970936，31171033)

Expression of a Chaining miR-shRNA for Simultaneous Knockdown of srGAP Family Members

 MA  Yue, DAI  Yun-Kai, CHEN  Keng, MI  Ya-Jing, JIN  Wei-Lin   

  1. (School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China)
  • Received:2011-03-20 Online:2012-05-28 Published:2012-05-28

摘要： 为了解析不同srGAP蛋白在神经元发育中的补偿效应，构建了一种同时靶向srGAP家族成员srGAP1~3的串联短发夹RNA表达系统.通过设计，合成多个寡核苷酸，利用微小RNA表达载体构建相应srGAP基因的短发夹RNA表达质粒，并利用蛋白免疫印迹法筛选敲减作用明显的靶向不同srGAP基因的短发夹RNA，然后，将其串联到同一载体而构建成可以同时靶向srGAP家族的短发夹RNA串联重组质粒.结果表明，筛选得到了分别针对srGAP1~3的短发夹RNA J17、J24和J33，并构建出能够同时靶向srGAP1~3基因的串联短发夹RNA  J1+2+3.

关键词: srGAP酶激活蛋白, 短发夹核糖核酸, 核糖核酸干扰, 蛋白免疫印迹

Abstract: To resolve the partial compensation effect of the different srGAPs in neuronal development, a miR-shRNA expression system was constructed which can down regulate srGAP family members srGAP1—3 simultaneously. Multiple miRshRNA oligos targeting different srGAPs were designed, synthesized and then inserted into the miRNA expression vector to generate miRshRNA against srGAP1—3 respectively. Western blot was performed to detect the knockdown effect of different miR-shRNAs, the three miRshRNA with the most obvious knockdown effect to srGAP1—3 respectively were selected. Then the miR-shRNAs were chained together in one vector to generate a chaining miRshRNA expression vector. The results show that the effective miRshRNA against corresponding srGAP1—3 are generated and named J17, J24 and J33. Furthermore, the chaining miR-shRNA J1+2+3 against srGAP1—3 is obtained.

Key words: Slit-Robo GTPase-activating protein (srGAP), short hairpin RNA (shRNA), RNA interference, Western blot

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