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Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China
Department of Pathophysiology, Basic Medical College, Zhengzhou University, Zhengzhou 450001, China
* Author to whom correspondence should be addressed.
Received: 8 April 2009; in revised form: 16 April 2009 / Accepted: 14 May 2009 / Published: 14 May 2009
Abstract: Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single- and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both single- and multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and β-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single- or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single- and multi-amiRNA expression vectors was comparable.
Keywords: artificial microRNA; expression vector; multiple genes; RNA interference
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MDPI and ACS Style
Hu, T.; Fu, Q.; Chen, P.; Ma, L.; Sin, O.; Guo, D. Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells. Int. J. Mol. Sci. 2009, 10, 2158-2168.
Hu T, Fu Q, Chen P, Ma L, Sin O, Guo D. Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells. International Journal of Molecular Sciences. 2009; 10(5):2158-2168.
Hu, Tao; Fu, Qiong; Chen, Ping; Ma, Li; Sin, Onsam; Guo, Deyin. 2009. "Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells." Int. J. Mol. Sci. 10, no. 5: 2158-2168.