Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (20): 3693-3696.doi: 10.3969/j.issn.1673-8225.2011.20.020

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Construction of a lentiviral vector for RNA interference targeting human toll-like receptor 4 gene

Ruan Jing1, Wang Xu1, Li Yu-sheng1, Liu Ai-hua2, Jiang Yong1   

  1. 1Department of Pathophysiology, Southern Medical University, Guangzhou  510515, Guangdong Province, China
    2Department of Respiration, Nanfang Hospital of Southern Medical University, Guangzhou  510515, Guangdong Province, China
  • Received:2010-12-29 Revised:2011-04-06 Online:2011-05-14 Published:2011-05-14
  • Contact: Jiang Yong, Doctoral supervisor, Professor, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, Guangdong Province, China jiang48231@163. com Correspondence to: Liu Ai-hua, Master’s supervisor, Associate professor, Department of Respiration, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China lah47158@yahoo. com.cn
  • About author:Ruan Jing★, Studying for master’s degree, Department of Pathophysiology, Southern Medical University, Guangzhou 510515, Guangdong Province, China ruanjing850617@ 163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81030055*; the United Foundation of the National Natural Science Foundation and the Natural Science Foundation of Guangdong Province, No. U0632004*; Development Plan for Cheung Kong Scholars and Innovative Teams, No. IRT0731*; the National 973
    Program of China, No. 2010CB529704*;Special Foundation for Doctors in High Education, No. 20069981001*; Science and Technology Program of Guangzhou City, No. 2007J1-C0301*

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Abstract:

BACKGROUND: As the most significant receptor mediating endotoxin or lipopolysaccharide response, toll-like receptor 4 (TLR4) plays a key role in the signaling pathway of inflammation induced by endotoxin.
OBJECTIVE: To construct a lentiviral RNAi vector that is capable of knocking down TLR4 gene in human umbilical vein endothelial cells (HUVECs).
METHODS: Short hairpin RNA (shRNA) sequence targeting Human TLR4 gene was designed using the Invitrogen online designing software. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were cloned into pENTRTM/H1/TO vectors and the resulting entry clones were identified by sequencing. Then, a recombinant reaction was performed to transfer the RNAi cassette from the entry plasmids into pLenti4/BLOCK-iTTM-DEST vectors to create the expression clone. Recombinant lentivirus was harvested from 293FT cells contransfected with the expression plasmids and the ViraPowerTM packing Mix. HUVEC cells were infected with the recombinant lentivirus and TLR4 expression in these cells was subsequently detected by western blot.
RESULTS AND CONCLUSION: Recombinant lentivirus expressing shRNA targeting the TLR4 gene was successfully constructed with the titer of 8.7×106 U/ml. Western blot result showed that the expression of TLR4 in lentivirus-infected HUVEC cells was significantly lower than that in the control cells. The lentiviral RNAi vector with the capability of knocking down TLR4 gene in HUVEC cells has been successfully constructed, providing a basis for further study on the relationship between inflammation and TLR4 gene.

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