Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (20): 3709-3713.doi: 10.3969/j.issn.1673-8225.2010.20.023

Previous Articles     Next Articles

Construction and identification of lentiviral vector targeting human bcl-2 and cyclin D1 gene

Wu Xiao-san1, Zhang Chun-lin1, Zhao Yao1, Gao Tian-tian2, Sun Jiao1, Zeng Bing-fang1   

  1. 1Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai  200233, China;
    2Department of Orthopaedics, Eighth People’s Hospital of Shanghai, Shanghai  200235, China
  • Online:2010-05-14 Published:2010-05-14
  • Contact: Zhang Chun-lin, Associate chief physician, Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China
  • About author:Wu Xiao-san, Studying for master’s degree, Physician, Department of Orthopaedics, Sixth People’s Hospital of Shanghai Jiao Tong University, Shanghai 200233, China Xiaosan96@live.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30700850*

PDF

341

Abstract:

BACKGROUND: Studies concerning osteosarcoma drug resistance are limited for single gene or passway. However, dual-channel blocker of apoptosis and cell cycle regulation may reverse drug resistance in osteosarcoma.

OBJECTIVE: To construct an effective bcl-2, cyclin D1 specific siRNA lentiviral vector and transfect it into drug-resistant osteosarcoma cell lines of osteosarcoma, and to discuss the reversal of drug resistance.

METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. Bcl-2 and cyclin D1 genes were cloned into the site of pSIH1-H1-copGFP shRNA vector to construct the pSIH1-H1-copGFP-bcl-2-siRNA and pSIH1-H1-copGFP-cyclinD1-siRNA. The cloned lentiviral plasmids and the packaging plasmids system were transfected into 293T cells. The supernatant was collected and the titer and infection efficiency of the recombinant lentivirus were determined.

RESULTS AND CONCLUSION: The ligation of four pairs of bcl-2 and cyclin D1 specific siRNAs to the double digested lentiviral pSIH1-H1-copGFP shRNA vector were successful. The titer of concentrated virus was 1.14×104 ifu/μL in the supernatant of the infected cells. The highest interference efficiency of bcl-2, cyclin D1 genes was respectively 88% and 87% detected by real-time fluorescence quantitative PCR. The results demonstrated an effective bcl-2, cyclin D1 specific siRNA lentiviral vector has been constructed by the application of siRNA technique.

CLC Number: