通过慢病毒载体构建SSA/Ro52稳定基因沉默细胞系

Establishment of Stable SSA/Ro52 Gene Silencing Cell Lines by Lentiviral Vectors

目的利用慢病毒载体持续表达RNA干扰序列抑制基因表达,建立SSA/Ro52稳定基因沉默细胞系,为进一步研究SSA/Ro52的功能提供有效工具。方法 4条SSA/Ro52特异序列(克隆1为TGGCATGGTCTCCTTCTACAA,克隆2为CTGCCTTCTTTATGGGACTTA,克隆9为TGAG从GTTGGAAGTGGAAAT,克隆1 1为AGTTATCCTATGGTCCTGGGT)和无关对照序列克隆至慢病毒载体pLKO-puro,表达后可形成小发卡结RNA(small hair-pin,shRNA),通过RNA干扰机制抑制特异基因表达。表达SSA/Ro52特异序列的克隆1、2、9、11以及表达无关序列的对照克隆(scramble,S)分别与包装载体pGag-Pol和pVSV-G共同转染293T细胞,收集含病毒颗粒的培养液。用表达SSA/Ro52特异序列和无关对照序列的病毒颗粒感染Hela细胞,抗生素筛选,建立稳定细胞系。以GAPDH为内对照定量实时PCR检测SSA/Ro52 mRNA的表达。免疫印记检测SSA/Ro52蛋白,扫描SSA/Ro52条带与β-actin条带相比为SSA/Ro52蛋白的相对表达水平。克隆1、2、9、11转染细胞中SSA/Ro52的表达水平与对照克隆S的比值为基因表达的抑制程度。结果慢病毒颗粒可以有效感染Hela细胞,含shRNA表达框架的病毒序列可以整合至细胞基因组,形成稳定细胞系。克隆1、2、9、1 1转染细胞在抗生素筛选3 d后,其SSA/Ro52 mRNA表达水平分别是克隆S转染细胞的25.5%、31.2%、13.0%和77.2%;而上述克隆转染细胞SSA/Ro52蛋白表达水平是对照的5%、50%、10%和80%。培养4周后上述克隆SSA/Ro52蛋白表达水平没有明显变化。但用干扰素α刺激细胞后,SSA/Ro52在对照克隆转染细胞中表达增加,而克隆1、2、9、11转染细胞SSA/Ro52的表达是对照的30%、70%、5%和100%。结论克隆1、2、9可以有效抑制SSA/Ro52的表达,通过慢病毒载体转染细胞建立了SSA/Ro52稳定基因静默细胞系。在SSA/Ro52表达被上调时,克隆9仍然可以有效抑制SSA/Ro52的表达,为进一步研究SSA/Ro52的功能打下了基础。

Objective ToestablishthestableSSA/Ro52genesilencingcelllinesbylentiviralvestorsexpressing RNA interference molecule which should be powerful tools facilitating the study of SSA/Ro52 function. Methods Four specific sequences of SSA/Ro52 （clone 1 TGGCATGGTCTCCTTCTACAA;clone 2 CTGCCT-TCTTTATGGGACTTA;clone 9 TGAGAAGTTGGAAGTGGAAAT;clone 1 1 AGTTATCCTATGGTCCTGG-GT）and unrelated control sequence S were cloned into lentiviral vector pLKO-puro,which could express short hairpin RNA （shRNA）and inhibit expression of specific gene by RNA interference mechanism. Clone1,2,9,11 which expressing SSA/Ro52 specific sequences and control clone S were packed with pGag-Pol and pVSV-G respectively,then 293 T cells were co-transfected. Culture medium containing virus particles were collected. The viral particles which expressing specific sequences SSA/Ro52 and unrelated control sequences were used to infected Hela cells,after antibiotic selection,stable cell lines were established. We used GAPDH as an internal control in real-time PCR for the detecting of SSA/Ro52 mRNA expression. Immunoblot was performed to detect SSA/Ro52 protein expression. The degree of gene expression inhibition was represented by the ratio of SSA/Ro52 expression level in Clone1,2,9,11 transfected cells and that in cloneS.Results LentiviruseffectivelyinfectedHelacells,thevectorDNAcontainingshRNAexpressing cassette were integrated into the cellullar gemone and stable cell lines were established. After antibiotics screening for 3 days,compared to transfected cells,the control clone S,the mRNA level of SSA/Ro52 of the transfected cells Clone 1,2,9,11 were 25. 5%,31. 2%,13. 0% and 77. 2%,respectively. While the protein level of SSA/Ro52 in the experimental group were 5%,50%,10%and 80%. After 4 weeks of culture,SSA/Ro52 protein expression did not change significantly. After stimulated by interferonα（IFN-α）, the transfected cells with control clone expressed increased level of SSA/Ro52 protein;while the protein level ofSSA/Ro52intheexperimentalgroupwere30%,70%,5%and100%.Conclusions Lentiviralvector clone 1 ,2 and 9 can significantly inhibit the expression of SSA/Ro52 and the stable SSA/Ro52 gene silencing cell lines have been successfully established. When SSA/Ro52 is upregulated by INF-α,clone 9 can still inhibit the expression of SSA/Ro52 effectively and can be used as a tool to study the function of SSA/Ro52.