parp10基因RNA干扰有效靶点的筛选及验证  

Screening and Identification of the Effective Small Interfering RNA Sequence Targeting parp10 Gene

作　　者：于孟斌[1,2] 周晓巍[1] 李朝[1] 张莹莹[1] 杨予涛[1] 王荣[1] 黄培堂[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]防化指挥工程学院生物防护教研室,北京102205

出　　处：《生物技术通讯》2009年第2期169-172,共4页Letters in Biotechnology

基　　金：国家科技支撑计划(2006BAD06A01)

摘　　要：目的:合成并筛选有效抑制parp10表达的siRNA。方法:根据parp10 cDNA序列,设计并合成prap10基因潜在的RNA干扰(RNAi)片段,将合成的寡核苷酸序列构建到pEGFP-C1H1U6载体中;通过双萤光素酶试验、Western blotting筛选有效的干扰序列;进一步用G418对A549细胞进行耐受度筛选,确定最低耐受度;用G418溶液对转染RNAi重组质粒的A549细胞进行筛选,通过RT-PCR鉴定干扰效果。结果:针对617bp处所构建的RNAi载体能够抑制PARP10的表达,用浓度为400μg/mL G418的McCoy′s 5A培养基筛选转染后的细胞,获得了能够表达绿色荧光标签蛋白的A549细胞株,经RT-PCR检测发现,PARP10表达受到抑制。结论:获得了能够有效抑制PARP10表达的特异性小干扰RNA(siRNA),为进一步研究其生物学功能提供了条件。Objective： To construct the RNA interference（RNAi） vector targeting parplO gene and screen the most effective small interfering RNA（siRNA） sequence. Methods： According to parplO cDNA sequence, the RNAi fragments targeting parplO gene were designed and synthesized. The siRNA eukaryotic expression vector pEGFP-C1H1U6-siRNA were constructed. Western blotting, dual-luciferase reporter assay and RT-PCR were carried out for the identification of integration of RNAi vector on mRNA and protein levels. Results： The specific siRNA vectors were constructed successfully, and the knockdown effect of siRNA at the site of 617 bp was confirmed. By the screening medium with 400 μg/mL G418, A549 clones expressing green fluorescent protein were obtained, in which PARP10 expression was suppressed markedly. Conclusion： parplO gene expression can be suppressed markedly by siRNA in mammalian cell. Based on the results, the further study on the biological functions and mechanisms of parplO will be carried out.

关 键 词：RNA干扰 PARP10 G418 

分 类 号：Q78[生物学—分子生物学]