Construction of an artificial MicroRNA expression vector for simultaneous inhibition of multiple genes in mammalian cells.
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 beta-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.
Hu
Tao
T
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Fu
Qiong
Q
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Chen
Ping
P
Department of Pathophysiology, Basic Medical College, Zhengzhou University, Zhengzhou 450001, China.
Ma
Li
L
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Sin
Onsam
O
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Guo
Deyin
D
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, China.
D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D035683:MicroRNAs; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D001616:beta-Galactosidase