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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-timePCR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48h收获的病毒粒子的感染效率最高,并且在包装后的24h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shRNARNA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。
BACKGROUND: Lentivirus-mediated RNAi technology has been widely used in gene function research because of its specificity, high inhibition efficiency and lasting effect. The technology virus packaging, transfection, and shRNA sequence design will have an impact on the inhibition efficiency. Therefore, experiments using human Bax inhibitor 1 (BI-1) as the target gene RNA interference experiments to explore its influencing factors. OBJECTIVE: To find siRNA targeting human BI-1 gene by lentivirus Lentivirus mediated RNAi technology. DESIGN, TIME AND SETTING: A one-sample observation was performed at the Department of Medical Genetics, School of Basic Medicine, Peking University from September 2007 to December 2008. Materials: 293T cells, SH-SY5Y cells were laboratory-held; 4 shRNAs were designed using the web-based online tool provided by Ambion (www.ambion.com). METHODS: shRNA recombinant expression vector targeting different regions of human BI-1 gene with green fluorescent protein (EGFP) tag was constructed and then transfected into 293T cells with the packaging protein expression vector to be packaged into 4 kinds of shRNA virus particles. GFP expression by flow cytometry to explore the best packaging conditions. Real-time PCR was performed using β-actin mRNA as an internal control. The four recombinant viruses and the control virus supernatant were used to infect SH-SY5Y, and the knockdown efficiency of the endogenous BI-1 gene was detected to select the best RNAi efficient sequence. MAIN OUTCOME MEASURES: The rate of intracellular GFP expression by different packaging viruses. Results: The optimal packing ratio of pLentiLox3.7, rev, vsvg, rre and other 4 plasmid packaging systems was 2: 1: 1: 1. The virus particles harvested at 48h had the highest infection efficiency and were changed after 24h Will improve the packaging efficiency. ShRNARNA targeted to the human BI-1 gene at -2-17 nucleotides (initiation coding region) has the highest efficiency of interference. Can inhibit 40% of normal gene expression. Conclusion: The experiment found out the important influencing factors of Lentivirus-mediated RNAi packaging. Which laid the foundation for establishing a stable knock-down neural cell model of human BI-1 gene expression and studying the pathological study of neuronal apoptosis diseases involved in the abnormal expression of BI-1.