中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (23): 4307-4311.doi: 10.3969/j.issn.1673-8225.2011.23.029

• 干细胞转基因表达 transgenic expression in stem cells • 上一篇    下一篇

新型慢病毒载体的构建及其在人骨髓间充质干细胞基因转导中的应用

卢晓芳1，杜晶春1，李  磊2，赖文玉3，张秀明1，李伟强1，杨  霞2，项  鹏1，2   

  1. 1中山大学干细胞与组织工程中心，广东省广州市  510080
    2中山大学中山医学院生物化学教研室，广东省广州市  510080
    3中山大学附属第二医院儿科，广东省广州市  510120
  • 收稿日期:2011-02-17 修回日期:2011-05-01 出版日期:2011-06-04 发布日期:2011-06-04
  • 通讯作者: 项鹏，教授，博士生导师，中山大学干细胞与组织工程中心，广东省广州市 510080；中山大学中山医学院生物化学教研室，广东省广州市510080 xiangp@mail.sysu.edu.cn
  • 作者简介:卢晓芳★，女，1985年生，河南省濮阳市人，汉族，中山大学医学院在读硕士，主要从事转基因技术的研究。 Zdluxiaofang2003@163.com
  • 基金资助:

    国家自然科学基金青年基金(81000150)；广东省自然科学基金博士启动(9451008901002230)。

Construction of modular lentivector and its application in transduction of human mesenchymal stem cells

Lu Xiao-fang1, Du Jing-chun1, Li Lei2, Lai Wen-yu3, Zhang Xiu-ming1, Li Wei-qiang1, Yang Xia2, Xiang Peng1,2   

  1. 1Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
    2Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou  510080, Guangdong Province, China
    3Department of Pediatrics, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
  • Received:2011-02-17 Revised:2011-05-01 Online:2011-06-04 Published:2011-06-04
  • Contact: Xiang Peng, Professor, Doctoral supervisor, Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China xiangp@mail.sysu.edu.cn
  • About author:Lu Xiao-fang★, Studying for master’s degree, Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China zdluxiaofang2003@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81000150*; the Natural Science Foundation of Guangdong Province, No. 9451008901002230*

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400

被引次数

3

摘要：

背景：构建一个组成性表达某特定基因同时携带荧光报告基因和抗生素筛选基因的慢病毒载体目前未见报道。
目的：观察新型慢病毒载体pLVpuro/EF1α-PEDF-IRES-EGFP在人骨髓间充质干细胞基因转导中的表达。
方法：通过PCR在PEDF基因的两端加上attB位点，构建表达载体pLVpuro/EF1α-PEDF-IRES-EGFP，将表达载体与包装质粒(ViraPowerTM Lentiviral Packaging Mix)共转染293FT细胞。通过多次感染的方法将慢病毒载体导入人骨髓间充质干细胞，转导后第7天开始使用1~5 mg/L嘌呤霉素筛选5 d，得到表达PEDF和EGFP的人骨髓间充质干细胞，并进行Western、Elisa的鉴定分析。
结果与结论：经PCR和测序证实，慢病毒表达载体pLVpuro/EF1α-PEDF-IRES-EGFP构建成功；将其成功导入人骨髓间充质干细胞，经过筛选获得纯化的过表达PEDF基因的绿色荧光细胞群。

关键词: 多位点Gateway技术, 慢病毒, 人骨髓间充质干细胞, PEDF, EGFP

Abstract:

BACKGROUND: Construction of a lentiviral vector concurrently containing a specific gene, a fluorescent reporter and an antibiotic selection marker under the control of different constitutive promoters for gene overexpression studies has not been reported before.
OBJECTIVE: To construct the lentivector, pLVpuro/EF1α-PEDF-IRES-EGFP, which containing the elements of PEDF and EGFP gene driven by EF1α promoter and puromycin resistant gene driven by PGK promoter through multisite gateway technology and to transduce it into human mesenchymal stem cells (hMSCs) to detect the protein expression of PEDF gene.
METHODS: The PEDF cDNA was flanked with attB sites by PCR and then cloned into pDONRTM221 to generate pDown-PEDF by utilizing the BP recombination reaction. The entry clones, pUp-EF1α, pDown-PEDF, pTail-EGFP were then recombined into the destination vector pDEST-puromycin to construct expression lentiviral vector, pLVpuro/EF1α-PEDF-IRES-EGFP by LR recombination reaction. The lentiviral particles were prepared by transient co-transfection of the resulting vector and ViraPower™ Lentiviral Packaging Mix into 293FT cells using Lipofectamine 2000. The viral particles were harvested concentrated and used to infect hMSCs. 7 days after transduction, 1-5 mg/L puromycin was added to the culture and lasted for 5 days. Transduced hMSCs were purified and then analyzed by Western Blot and ELISA assay.
RESULTS AND CONCLUSION: In this study, the lentivector pLVpuro/EF1α-PEDF- IRES-EGFP was successfully constructed by multisite gateway technology and it was confirmed by PCR and gene sequencing and the hMSCs were successfully transduced by lentivirus. After antibiotic selection, pure infected cell population which expressed PEDF gene and EGFP were attained.

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