Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (32): 6036-6040.doi: 10.3969/j.issn.1673-8225.2011.32.033

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Construction and identification of lentiviral vector encoding shRNA against REST/NRSF

Li Hong-tu1, 2, Shi Ping3, Pang Xi-ning1   

  1. 1Department of Stem Cells and Reproductive Medicine, Key Laboratory of Cell Biology, Ministry of Public Health, China Medical University, Shenyang  110001, Liaoning Province, China
    2 Key Laboratory of Reproduction Health of Liaoning Province, Liaoning Province Research Institute of Family Planning, Shenyang  110031, Liaoning Province, China
    3Department of General Practice, the First Affiliated Hospital, China Medical University, Shenyang  110001, Liaoning Province, China
  • Received:2011-02-02 Revised:2011-03-16 Online:2011-08-06 Published:2011-08-06
  • Contact: Pang Xi-ning, Doctor, Professor, Department of Stem Cells and Reprodcective Medicine, Key Laboratory of Cell Biology, Ministry of Public Health, China Medical University, Shenyang 110001, Liaoning Province, China pxining@yahoo.com
  • About author:Li Hong-tu☆, Studying for doctorate, Associate researcher, Department of Stem Cells and Reproductive Medicine, Key Laboratory of Cell Biology, Ministry of Public Health, China Medical University, Shenyang 110001, Liaoning Province, China; Key Laboratory of Reproduction Health of Liaoning Province, Liaoning Province Research Institute of Family Planning, Shenyang 110031, Liaoning Province, China lhongtu@126.com Shi Ping, Professor, Department of General Practice, the First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China shiping57428@yahoo.com.cn Li Hong-tu and Shi Ping contributed equally to this paper.
  • Supported by:

    the Science and Technology Plan Project of Liaoning Science and Technology Bureau, No. 2010225034*; Shenyang Science and Technology Plan, No. 2009-090063*, 2011-F10-222-4-00*

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Abstract:

BACKGROUND: In recent years, many studies showed that respressor element1(RE-1) 1-silencing transcription factor /neurons restrictive silence factor (REST/NRSF) could negative control neurons and islet cell differentiation related gene expression.
OBJECTIVE: To construct a lentiviral vector expressing shREST/NRSF.
METHODS: Four groups of shRNA sequences specifically targeting the REST/NRSF gene were designed and synthesized and cloned into the pFU-GW-RNAi vector. The obtained lentiviral vector containing shREST/NRSF was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector L-shREST/NRSF, pHelper1.0 and pHelper2.0.The titer of virus was tested according to the expression level of GFP. And preliminary observations on the situation of transfected rat bone marrow mesenchymal stem cells. Real time PCR was employed to assess the gene silencing efficacy of these recombinants.
RESULTS AND CONCLUSION: PCR and DNA sequencing demonstrated that the constructed lentivirus vector L-shNRSE/RE-1 produced REST/NRSF shRNA. And it could be stably transfected to rat bone marrow mesenchymal stem cells, there was the infection efficiency of almost 100%. All of these four shRNAs could achieve gene knock down effect, and 3# shRNA had the most significant gene silence effect among them. The lentivirus vector of shREST/NRSF is constructed successfully.

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