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

被引:19
|
作者
Hu, Tao [1 ,2 ]
Fu, Qiong [1 ,2 ]
Chen, Ping [3 ]
Ma, Li [1 ,2 ]
Sin, Onsam [1 ,2 ]
Guo, Deyin [1 ,2 ]
机构
[1] Wuhan Univ, State Key Lab Virol, Wuhan 430072, Peoples R China
[2] Wuhan Univ, Modern Virol Res Ctr, Coll Life Sci, Wuhan 430072, Peoples R China
[3] Zhengzhou Univ, Dept Pathophysiol, Basic Med Coll, Zhengzhou 450001, Peoples R China
来源
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES | 2009年 / 10卷 / 05期
关键词
artificial microRNA; expression vector; multiple genes; RNA interference; SHORT HAIRPIN RNAS; POLYMERASE-II; DROSHA-DGCR8; COMPLEX; NUCLEAR EXPORT; MESSENGER-RNAS; INTERFERENCE; PRECURSORS; GENERATION; KNOCKDOWN; CLONING;
D O I
10.3390/ijms10052158
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:2158 / 2168
页数:11
相关论文
共 50 条
  • [11] Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector
    Tao Hu
    Qiong Fu
    Ping Chen
    Ke Zhang
    Deyin Guo
    Biotechnology Letters, 2009, 31
  • [12] Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector
    Hu, Tao
    Fu, Qiong
    Chen, Ping
    Zhang, Ke
    Guo, Deyin
    BIOTECHNOLOGY LETTERS, 2009, 31 (03) : 353 - 359
  • [13] Construction and application of a novel hepatocyte-directed vector to simultaneous knockdown and overexpression of multiple genes
    Geng, Jianlin
    Wei, Haiming
    Sun, Rui
    Tian, Zhigang
    LIVER INTERNATIONAL, 2014, 34 (07) : E246 - E256
  • [14] Mutated Polyadenylation Signals for Controlling Expression Levels of Multiple Genes in Mammalian Cells
    Yang, Yuansheng
    Mariati
    Ho, Steven C. L.
    Yap, Miranda G. S.
    BIOTECHNOLOGY AND BIOENGINEERING, 2009, 102 (04) : 1152 - 1160
  • [15] The construction and characterization of a novel adenovirus vector of artificial microRNA targeting EGFR
    Chen, Ping
    Zhang, Shou-De
    Lin, Yi
    Cao, Jiang
    Chen, Jia
    Yang, Bei-Bei
    INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY, 2019, 12 (06): : 1968 - 1974
  • [16] A retroviral vector for siRNA expression in mammalian cells
    Gro Leite Størvold
    Elisabet Gjernes
    Hanne Arenberg Askautrud
    Anne-Lise Børresen-Dale
    Charles M. Perou
    Eirik Frengen
    Molecular Biotechnology, 2007, 35 : 275 - 282
  • [17] A retroviral vector for siRNA expression in mammalian cells
    Storvold, Gro Leite
    Gjernes, Elisabet
    Askautrud, Hanne Arenberg
    Borresen-Dale, Anne-Lise
    Perou, Charles M.
    Frengen, Eirik
    MOLECULAR BIOTECHNOLOGY, 2007, 35 (03) : 275 - 282
  • [18] Construction of a mammalian cell expression vector pAcGFP-FasL and its expression in bovine follicular granulosa cells
    Yang, RunJun
    Huang, Meng
    Li, JunYa
    Zhao, ZhiHui
    Xu, ShangZhong
    AFRICAN JOURNAL OF BIOTECHNOLOGY, 2011, 10 (59): : 12819 - 12828
  • [19] DESIGN OF A RETROVIRUS-DERIVED VECTOR FOR EXPRESSION AND TRANSDUCTION OF EXOGENOUS GENES IN MAMMALIAN-CELLS
    PERKINS, AS
    KIRSCHMEIER, PT
    GATTONICELLI, S
    WEINSTEIN, IB
    MOLECULAR AND CELLULAR BIOLOGY, 1983, 3 (06) : 1123 - 1132
  • [20] Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells
    Medley, CD
    Drake, TJ
    Tomasini, JM
    Rogers, RJ
    Tan, WH
    ANALYTICAL CHEMISTRY, 2005, 77 (15) : 4713 - 4718