RACK1干扰质粒的构建及其干扰效率的鉴定

Construction and Identification of Interference Plasmid Targeting in RACK1

目的构建高效针对RACK1(Gnb2l1基因)的干扰质粒,为进一步研究RACK1功能构建有效平台。方法采用M-folder生物软件选择2个Gnb2l1干扰位点,根据位点序列合成2个干扰片段及1个阴性对照片段,定向克隆入pGenesil-1干扰载体并测序验证。将干扰及对照质粒分别转染至NIH3T3细胞后,实时定量RT-PCR鉴定干扰效率。结果实时定量RT-PCR结果显示,干扰质粒转染组的Gnb2l1mRNA量分别为未转染组的58%和7%,阴性对照质粒转染组与未转染组一致,提示干扰质粒pGene-sil-1/Gnb2l1-Ⅱ干扰效率达到90%以上。结论成功构建并筛选到具有显著干扰效率的干扰质粒,为进一步研究RACK1及其伴侣分子的功能联系奠定了基础。

Objective To construct highly efficient interference plasmid targeting in RACK1 （Mouse Gnb2l1 gene）, and make a basis for studying the function of RACK1. Methods Two interference sites of mouse Gnb2l1 gene were selected by using M-folder bio-software, and two interference fragments according to the selected sequences and one negative-control fragment were synthesized, then they were cloned into the pGenesil-1 plasmid. After the plasmids were extracted and sequenced, they were subsequently transfected into NIH3T3 cells. The interference efficiency of the plasmid on the target gene was detected by RT-PCR. Results RT-PCR showed that the level of Gnb2l1 mRNA in NIH3T3 cells contained interference plasmids was 58% and 7% of no-load group, respectively, and mRNA expression in negative-control plasmid group and no-load group were coincidence. It was suggested that the interference efficiency was above 90% due to pGenesil-1/ Gnb2l1-Ⅱ interference plasmid. Conclusion The highly efficient interference plasmid against Mice Gnb2l1 gene had been constructed and identified successfully, which may provide a basis for studying the functional connection between RACK1 and partner moleculars.