靶向srGAP家族的串联短发夹RNA表达

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

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.

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, synthe- sized 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 srGAPl-3 are generated and named J17, J24 and J33. Furthermore, the chaining miR-shRNA J1＋2＋3 against srGAP1-3 is obtained.