High-Throughput Screening of Effective siRNAs Using Luciferase-Linked Chimeric mRNA

The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences) of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

利用小干扰RNA（siRNA）敲低基因表达，有望成为治疗多种疾病的潜在策略。为规避siRNA的毒性，已有研究提出选用转录活性更低的H1 RNA聚合酶III（pol III）启动子，而非U6启动子，来驱动siRNA的表达。由于当前的计算机预测程序难以获得理想结果，若要鉴定高效的siRNA序列，则需要开展大规模筛选实验。本研究以CCR5基因沉默为模型，探究一种快速高效的siRNA筛选方法。我们构建了嵌合荧光素酶-CCR5基因，用于siRNA文库的高通量筛选。在对约900个短发夹RNA（shRNA）克隆完成筛选后，共鉴定得到12条有效siRNA序列。序列分析结果显示，这12条序列中的绝大多数（11条）均未被现有siRNA预测算法所覆盖。我们通过将对应siRNA构建为慢病毒载体并感染细胞，证实了这些鉴定得到的siRNA可显著抑制T淋巴细胞细胞系以及原代T细胞中的CCR5表达。该CCR5表达抑制作用可使细胞免受R5型HIV-1 JRCSF株的感染。本研究结果表明，该高通量筛选方法可在低表达水平下高效鉴定出能够抑制靶基因的siRNA序列。