中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (32): 6036-6040.doi: 10.3969/j.issn.1673-8225.2011.32.033

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

REST/NRSF基因shRNA慢病毒载体的构建与鉴定

李宏图1，2，施  萍3，庞希宁1   

  1. 1中国医科大学细胞生物学卫生部重点实验室干细胞与再生医学研究室，辽宁省沈阳市  110001
    2辽宁省计划生育科学研究院辽宁省生殖医学重点实验室，辽宁省沈阳市  110031
    3中国医科大学附属第一医院全科医学教研室，辽宁省沈阳市  110001
  • 收稿日期:2011-02-02 修回日期:2011-03-16 出版日期:2011-08-06 发布日期:2011-08-06
  • 通讯作者: 庞希宁，教授，博士，博士生导师，中国医科大学细胞生物学卫生部重点实验室干细胞与再生医学研究室，辽宁省沈阳市110001 pxining@yahoo.com
  • 作者简介:李宏图☆，男，1977年生，辽宁省岫岩满族自治县人，满族，中国医科大学在读博士，副研究员，主要从事干细胞和生殖生物学研究。 lhongtu@126.com 并列第一作者：施萍，女，1957年生，辽宁省沈阳市人，汉族，教授，主要从事应用干细胞技术治疗糖尿病及糖尿病足的研究。 shiping57428@yahoo.com.cn
  • 基金资助:

    辽宁省科学技术厅科学技术计划项目(2010225034)；沈阳市科学技术计划(2009-090063, 2011-F10-222-4-00)。 

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*

PDF

387

被引次数

5

摘要：

背景：神经元限制性沉默因子(REST/NRSF)能负向调控神经元及胰岛细胞分化相关基因的表达。
目的：构建并筛选能高效沉默大鼠REST/NRSF基因的shRNA慢病毒载体。
方法：针对REST/NRSF基因设计4组特异性siRNA靶点，合成靶序列的寡核苷酸序列，退火形成双链DNA，与经Hpa Ⅰ 和Xho Ⅰ双酶切后的pFU-GW-RNAi载体连接产生L-shREST/NRSF慢病毒载体，PCR筛选阳性克隆，测序鉴定。包装产生慢病毒颗粒，随后将其感染大鼠骨髓间充质干细胞，采用 Real-time PCR方法检测靶基因在 mRNA 水平的沉默效率。
结果与结论：PCR和测序证实，构建出了REST/NRSF shRNA的慢病毒载体L-shREST/NRSF，并能稳定转染大鼠间充质干细胞，感染效率达100%。4组shRNA序列均有基因沉默效果，并以第3组shRN序列效果最为明显。结果表明，该慢病毒表达载体能够在细胞水平有效沉默靶基因。

关键词: 慢病毒载体, REST/NRSF, shRNA, 基因, 骨髓间充质干细胞

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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