An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level.
An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level.
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DOI：
10.12688/f1000research.3-60.v1
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发表时间：
2014-01-01
期刊：
影响因子：
--
通讯作者：
Borisy, Gary G
中科院分区：
文献类型：
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作者：
Kojima, Shin-Ichiro;Borisy, Gary G
RNA干扰（RNAi）被广泛用于以特定方式抑制基因表达。RNAi的疗效主要取决于小干扰RNA（siRNA）相对于靶mRNA的序列。尽管已经开发了几种用于siRNA设计的算法，但是仍然难以从多个候选者中选择真正有效的siRNA。在这篇文章中，我们报告了一种基于图像的，定量的，比率荧光报告基因检测的发展，以评估在单细胞水平的RNAi的疗效。使用两种荧光报告构建体。一个表达候选小发夹RNA（shRNA）以及增强的绿色荧光蛋白（EGFP）;另一个表达插入表达红色荧光蛋白（DsRed或mCherry）的盒中的19-nt靶序列。候选shRNA的有效性被评估为它敲低红色荧光蛋白表达的程度。因此，红色与绿色荧光强度比（相对于对照适当标准化）被用作在单个细胞水平上定量siRNA功效的读数。我们测试了这种双重荧光测定，并将预测结果与三种不同基因（波形蛋白、核纤层蛋白A/C和Arp 3）和二十种不同shRNA的实际内源性敲低水平进行了比较。对于每个基因，我们的测定成功地预测了有效RNAi的靶序列。为了进一步促进RNAi功效的测试，我们开发了一种用于构建shRNA和红色荧光报告质粒的阴性选择标记（ccdB）方法，该方法允许我们直接从转化的细菌中纯化这些质粒，而不需要菌落选择和DNA测序验证。
RNA interference (RNAi) is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA) in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA) together with an enhanced green fluorescent protein (EGFP); the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry). Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls) is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3) and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker ( ccdB) method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.