TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents
TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents
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DOI：
10.3791/53879
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发表时间：
2016-06-01
影响因子：
1.2
通讯作者：
Nashimoto, Masayuki
中科院分区：
文献类型：
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作者：
Haino, Arisa;Ishikawa, Tatsuya;Nashimoto, Masayuki
TRUE基因沉默（又称tRNase Z(L)- - -利用有效的基因沉默）是一种RNA定向基因沉默技术，它利用人工小向导RNA （sgRNA）引导tRNA 3'加工核糖核酸内切酶tRNase Z(L)识别目标RNA。sgRNAs无需任何转染试剂即可被细胞吸收，并可下调其靶RNA水平和/或诱导人类癌细胞凋亡。我们筛选了一个包含156个七聚体型sgRNA的sgRNA文库，研究其对人类骨髓瘤和白血病细胞活力的影响，发现其中20个sgRNA可以在至少一种癌细胞系中有效诱导细胞凋亡。在这里，我们提出了一种筛选七聚体型sgRNA文库的方案，用于潜在的治疗血癌的药物。该方案包括如何构建sgRNA文库，如何评估每种sgRNA对细胞活力的影响，以及如何通过流式细胞术进一步评估有效的sgRNA。通过筛选由16,384个七聚体组成的全尺寸sgRNA文库，预计将获得约2,000个命中点。
TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.