CAJ | 학술논문

背景: 慢病毒介导的RNAi技术以其专一性、抑制效率高、作用持久等优点已广泛应用于基因功能研究中.该技术病毒包装、转染、以及shRNA序列设计都会对抑制效率产生影响.因此实验以人的Bax抑制子1 (BI-1)为目的基因进行RNA干扰实验来探讨其影响因素.目的:为了利用慢病毒Lentivirus介导的RNAi技术寻找抑制人BI-1基因表达的siRNA.设计、时间及地点:单一样本观察,于2007-09/2008-12在北京大学医学部基础医学院医学遗传学系完成.材料:293T细胞、SH-SY5Y细胞为实验室保存;用Ambion公司(www.ambion.com)提供的网络在线工具设计了4个shRNA.方法:构建带有绿色荧光蛋白(EGFP)标签的、针对人BI-1基因不同区域设计的shRNA重组表达载体,然后与包装蛋白表达载体共转染293T细胞以包装成4种shRNA病毒粒子.通过流式细胞仪检测GFP的表达情况来摸索最佳包装条件.Real-time PCR以β-肌动蛋白mRNA被用作内参,将4种重组病毒和对照组病毒上清液侵染SH-SY5Y,检测内源性BI-1基因的敲低效率,筛选到最佳RNAi有效序列.主要观察指标:被不同包装病毒侵染的细胞内GFP的表达率.结果:pLentiLox3.7、rev、vsvg、rre等4质粒包装系统的最佳包装比例为2∶1:1∶1,包装48 h收获的病毒粒子的感染效率最高,并且在包装后的24 h之后换液会提高包装效率.靶定到人BI-1基因-2-17核苷酸(起始编码区域)的shRNA RNA干扰效率最高.能够抑制40%正常基因表达.结论:实验摸索出Lentivirus介导的RNAi技术病毒包装的重要影响因素.为建立稳定的人BI-1基因表达敲低的神经细胞模型和研究BI-1异常表达参与的神经元凋亡疾病的病理研究打下基础.
배경: 만병독개도적RNAi기술이기전일성、억제효솔고、작용지구등우점이엄범응용우기인공능연구중.해기술병독포장、전염、이급shRNA서렬설계도회대억제효솔산생영향.인차실험이인적Bax억제자1 (BI-1)위목적기인진행RNA간우실험래탐토기영향인소.목적:위료이용만병독Lentivirus개도적RNAi기술심조억제인BI-1기인표체적siRNA.설계、시간급지점:단일양본관찰,우2007-09/2008-12재북경대학의학부기출의학원의학유전학계완성.재료:293T세포、SH-SY5Y세포위실험실보존;용Ambion공사(www.ambion.com)제공적망락재선공구설계료4개shRNA.방법:구건대유록색형광단백(EGFP)표첨적、침대인BI-1기인불동구역설계적shRNA중조표체재체,연후여포장단백표체재체공전염293T세포이포장성4충shRNA병독입자.통과류식세포의검측GFP적표체정황래모색최가포장조건.Real-time PCR이β-기동단백mRNA피용작내삼,장4충중조병독화대조조병독상청액침염SH-SY5Y,검측내원성BI-1기인적고저효솔,사선도최가RNAi유효서렬.주요관찰지표:피불동포장병독침염적세포내GFP적표체솔.결과:pLentiLox3.7、rev、vsvg、rre등4질립포장계통적최가포장비례위2∶1:1∶1,포장48 h수획적병독입자적감염효솔최고,병차재포장후적24 h지후환액회제고포장효솔.파정도인BI-1기인-2-17핵감산(기시편마구역)적shRNA RNA간우효솔최고.능구억제40%정상기인표체.결론:실험모색출Lentivirus개도적RNAi기술병독포장적중요영향인소.위건립은정적인BI-1기인표체고저적신경세포모형화연구BI-1이상표체삼여적신경원조망질병적병리연구타하기출.
BACKGROUND: Lentivirus-mediated RNA interference technology has been widely used in the study of gene function because of its specificity, high inhibition efficiency and persistent effects. Virus packaging, transfection, as well as small hairpin RNA (shRNA)-coding sequence could affect the inhibition efficiency. Therefore, impact factors of RNA interference for human Bax inhibitor 1 (BI-1) was studied in this experiment.OBJECTIVE: To find the valid small interference RNA (siRNA) targeting human BI-1 gene by Lentivirus.DESIGN, TIME AND SETTING: Single sample observation was completed in Department of Medical Genetics, Basic Medical College, Peking University Health Science Center from September 2007 to December 2008.MATERIALS: 293T cell and SH-SY5Y cells were preserved in this laboratory; 4 shRNAs were designed through a use of online design tool by Ambion (www.ambion.com). METHODS: Several recombinant plasmids which expressed shRNAs targeting different regions of the BI-1 gene and labeled enhanced green fluorescent protein as a fusion gene were constructed. Four types of shRNA-expressing virions were obtained by cotransfection of shRNA-expressing plasmid and package protein-expressing plasmids into 293T cells. Green fluorescent protein expression was determined using flow cytometry to search the optimal package conditions. Real-time PCR and beta actin mRNA was used as an internal control, four types of recombinant viral supernatant and control viral supernatant were added on the SH-SY5Y cells, knock-down efficacies of the endogenous BI-1 gene were determined, and the RNA interference effective sequences were optimized. MAIN OUTCOME MEASURES: Intracellular green fluorescent protein expression rate of the cells infected with different packaging virus. RESULTS: The optimal infective packaging virus could be obtained when the ratio of plasmids pLentiLox3.7, REV, VSVG and RRE was 2:1:1:1 and the supernatants were collected 48 hours after transfection. Replacement of culture media 24 hours after transfection could increase the infection efficacy of the virions. shRNA targeting-2-17nt (start coding region) of BI-1 gene was most valid interference efficiency and could decrease the gene expression to 40%. CONCLUSION: Influencing factors of Lentivirus-mediated RNA interference technology virus packages were investigated in this study, which could be a foundation for constructing stable BI-1 knocked down neuronal cell model and for studying the abnormal expression of BI-1 related to the neuron apoptosis disease.