Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo.
Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo.
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DOI：
10.3389/neuro.17.001.2009
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发表时间：
2009
影响因子：
4.3
通讯作者：
Cline HT
中科院分区：
文献类型：
--
作者：
Chen CM;Chiu SL;Shen W;Cline HT
RNA干扰(RNAi)是一种进化保守的序列特异性基因沉默机制。最近我们对RNAi机制的了解取得了进展，通过将短发夹状RNA(ShRNA)引入许多系统的细胞中来减少蛋白质的表达，然而，RNAi介导的蛋白质敲除的效果可能是非常不同的，特别是在完整的动物中，这限制了它的应用。我们构建了适应性分子工具pSilencer(PSI)和pReporter(Pre)结构，以评估不同启动子、shRNA结构和RNA诱导沉默复合体中的关键酶Ago2的过度表达对RNAi效率的影响。通过比较RNAi靶标GFP和非靶标mCherry的荧光强度水平，评估了培养细胞和完整动物中RNAi敲除的程度。人Ago2和shRNA的共表达显著提高了GFP在细胞系和完整非洲爪哇蝌蚪神经元中的敲除效率。单独的人H1和U6启动子或带有增强子元件的U6启动子在驱动GFP敲除方面同样有效。来自microRNA-30设计的shRNA(ShRNAmir30)提高了GFP基因敲除的效率。用shRNA表达含有Ago2的PSI可以提高内源性神经元蛋白-AMPA受体的GluR2亚单位的击倒效率，该亚单位通过记录AMPA受体介导的自发突触电流在非洲爪哇中枢神经系统神经元上获得功能。我们的数据表明，Ago2和shRNA的共同表达是一种简单的增强完整动物RNAi的方法。虽然在非洲爪哇和斑马鱼中，吗啡反义基因敲除是有效的，但RNAi方法的一个主要优势是，通过使用细胞类型特异性和可调控的PolII启动子来驱动shRNA和Ago2，有可能对蛋白质基因敲除进行空间和时间控制。这应该会扩大RNAi在研究完整脑回路基因功能方面的应用。
RNA interference (RNAi) is an evolutionarily conserved mechanism for sequence-specific gene silencing. Recent advances in our understanding of RNAi machinery make it possible to reduce protein expression by introducing short hairpin RNA (shRNA) into cells of many systems, however, the efficacy of RNAi-mediated protein knockdown can be quite variable, especially in intact animals, and this limits its application. We built adaptable molecular tools, pSilencer (pSi) and pReporter (pRe) constructs, to evaluate the impact of different promoters, shRNA structures and overexpression of Ago2, the key enzyme in the RNA-induced silencing complex, on the efficiency of RNAi. The magnitude of RNAi knockdown was evaluated in cultured cells and intact animals by comparing fluorescence intensity levels of GFP, the RNAi target, relative to mCherry, which was not targeted. Co-expression of human Ago2 with shRNA significantly enhanced efficiency of GFP knockdown in cell lines and in neurons of intact Xenopus tadpoles. Human H1- and U6-promotors alone or the U6-promotor with an enhancer element were equally effective at driving GFP knockdown. shRNA derived from the microRNA-30 design (shRNAmir30) enhanced the efficiency of GFP knockdown. Expressing pSi containing Ago2 with shRNA increased knockdown efficiency of an endogenous neuronal protein, the GluR2 subunit of the AMPA receptor, functionally accessed by recording AMPA receptor-mediated spontaneous synaptic currents in Xenopus CNS neurons. Our data suggest that co-expression of Ago2 and shRNA is a simple method to enhance RNAi in intact animals. While morpholino antisense knockdown is effective in Xenopus and Zebrafish, a principle advantage of the RNAi method is the possibility of spatial and temporal control of protein knockdown by use of cell type specific and regulatable pol II promoters to drive shRNA and Ago2. This should extend the application of RNAi to study gene function of intact brain circuits.
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影响因子：
4.5
作者：
Cai, XZ;Hagedorn, CH;Cullen, BR
通讯作者：
Cullen, BR
影响因子：
12.4
作者：
Giering, Jeffery C.;Grimm, Dirk;Kay, Mark A.
通讯作者：
Kay, Mark A.
DOI：
10.1093/nass/48.1.191
发表时间：
2004-01-01
期刊：
Nucleic acids symposium series (2004)
影响因子：
--
作者：
Mikuma, Toshiyasu;Kawasaki, Hiroaki;Taira, Kazunari
通讯作者：
Taira, Kazunari
影响因子：
5.3
作者：
Anichtchik, Oleg;Diekmann, Heike;Rubinsztein, David C.
通讯作者：
Rubinsztein, David C.
DOI：
10.1073/pnas.0800803105
发表时间：
2008-07-08
影响因子：
11.1
作者：
Diederichs, Sven;Jung, Stephanie;Habert, Daniel A.
通讯作者：
Habert, Daniel A.