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Int. J. Mol. Sci. 2009, 10(5), 2158-2168; doi:10.3390/ijms10052158
Communication

Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells

1
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1
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2
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1
,
1
 and
1,*
1 State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China 2 Department of Pathophysiology, Basic Medical College, Zhengzhou University, Zhengzhou 450001, China
* Author to whom correspondence should be addressed.
Received: 8 April 2009 / Revised: 16 April 2009 / Accepted: 14 May 2009 / Published: 14 May 2009
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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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 artificial microRNA; expression vector; multiple genes; RNA interference
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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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.

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