A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector.
A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector.
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一种通过单一载体一步转染快速筛选有效 siRNA 靶位点的新型系统。
DOI：
10.1016/j.jbiotec.2011.05.004
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发表时间：
2011-09
影响因子：
4.1
通讯作者：
中科院分区：
文献类型：
--
作者：
SiRNA技术已被广泛应用于基因功能的研究和疾病治疗的研究。获得最有效的靶向特异性siRNA序列是RNAi应用面临的挑战之一。目前，选择和验证给定基因的最佳siRNA位点的过程，通常是基于Western印迹、Northern印迹或Q-PCR筛选，仍然是经验和耗时的。虽然很少有基于荧光的siRNA序列选择系统被证明是有效的，但快速有效地筛选siRNA靶点仍然是具有挑战性的。本文建立了一种快速有效的单载体系统筛选siRNA靶点的方法，该方法包括：(1)绿色荧光蛋白报告基因表达盒，随后是多克隆位点和插入靶序列的SV40 pA；(2)含有反向驱动的双POI III启动子的siRNA表达盒；(3)内部加载对照，mCherry报告基因。基于这种单载体系统的一步转染法，我们可以通过判断EGFP与mCherry的荧光强度比，以无偏见的方式快速筛选有效的、靶向的siRNA片段。这种新型载体系统的产生将促进siRNA在基础研究和疾病治疗中的应用。
siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.
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影响因子：
46.9
作者：
Dassie, Justin P.;Liu, Xiu-ying;Thomas, Gregory S.;Whitaker, Ryan M.;Thiel, Kristina W.;Stockdale, Katie R.;Meyerholz, David K.;McCaffrey, Anton P.;McNamara, James O., II;Giangrande, Paloma H.
通讯作者：
Giangrande, Paloma H.
影响因子：
14.9
作者：
Naito, Y;Yamada, T;Saigo, K
通讯作者：
Saigo, K
DOI：
--
发表时间：
2009-07
期刊：
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
影响因子：
--
作者：
Hui-jin Li;Weifeng Zhang;Dong-yang Wang;Rong-hua Li;Zong-li Qi;Jun-li Zhao;Hai-bin Xia
通讯作者：
Hui-jin Li;Weifeng Zhang;Dong-yang Wang;Rong-hua Li;Zong-li Qi;Jun-li Zhao;Hai-bin Xia
影响因子：
5.8
作者：
Luquan Wang;Forest Y. Mu
通讯作者：
Luquan Wang;Forest Y. Mu
RNA干扰HIV感染的细胞靶标的治疗潜力。
影响因子：
2.6
作者：
Zhang J;Wu YO;Xiao L;Li K;Chen LL;Sirois P
通讯作者：
Sirois P