BGC-823细胞中慢病毒途径针对HIF-1α基因的RNAi实验

目的：构建人HIF-1α基因的shRNA慢病毒载体、包装生产重组慢病毒颗粒,病毒途径高效感染胃癌细胞株BGC-823,并验证基因沉默效率。方法：在NCBI数据查找人HIF-1α基因序列,然后使用siRNA在线设计软件,设计针对基因CDS区的3条siRNA序列及Negative序列。根据设计好的siRNA序列设计双链互补的shRNA-Oligo DNA,退火形成双链后与线性化载体链接,构建shRNA重组表达载体。经由293TN细胞包装shRNA重组慢病毒颗粒,随后使用重组病毒感染BGC-823,通过荧光标记蛋白GFP确定感染效率后收集细胞样本,分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率。结果：shRNA重组表达载体测序结果与设计序列完全一致,包装病毒后滴度达到1×104 ifu/μL。慢病毒感染BGC-823细胞取得了极高的基因转导效率。mRNA检测结果显示,siRNA3对于对与目的基因的沉默效果最好,较阴性对照序列组相比较,mRNA的表达量下降了92%,Western blot检测的结果与mRNA检测结果完全相符合。结论：通过慢病毒途径,可以在BGC-823细胞中高效的进行HIF-1α基因的沉默。

RNAi of HIF-1 α Gene in BGC-823 Cells through Lentiviral Approach

Objective,To construct shRNA lentiviral vector targeted to human HIF-1α gene,to package and produce recombinant lentivirial particles,infect a gastric cancer cell line BGC-823 efficiently,and to verify the efficiency of gene silencing.Methods： the gene sequence of HIF-1α was searched in NCBI database,and online siRNA design software was used to design three siRNA sequences targeted gene CDS region and a Negative sequence.Double-stranded complementary Oligo DNA sequences for designed siRNA sequences were designed,and annealed to form double strands,and linked to linear vector to construct recombinant shRNA expression vector.293TN cells were used for shRNA lentiviral particle packaging,and the recombinant virus was used to infect BGC-823.Fluorescent marker GFP was used to determine the infection efficiency,and the cell sample was collected.Real-time PCR and Western blot were used to detect the silencing efficiency of target gene at mRNA and protein levels.Results：recombinant shRNA expression vector sequencing results are consistent with the design sequence,packaged virus titer was 1×104 ifu/μL.Infection of BGC-823 cells by lentivirus produced high gene transduction efficiency.Results of mRNA detection show that siRNA3 has the best silencing effect on target gene,and the mRNA expression was decreased by 92% compared with negative control group.Moreover,Western blotting results are completely consistent with the mRNA results.Conclusion：Lentivirus can inhibit the expression of HIF-1α gene in BGC-823 cells efficiently.