Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (42): 7830-7836.doi: 10.3969/j.issn.2095-4344.2012.42.008

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Expression of the retroviral vector of RNA interference for human estrogen receptor beta in human MG63 osteoblast-like cells

Wang Yu-xiang, Zhang Hong-qi, Guo Chao-feng, Tang Ming-xing, Liu Shao-hua, Deng Ang, Gao Qi-le, Liu Jin-yang, Wu Jian-huang   

  1. Department of Spinal Surgery, Xiangya Spinal Surgery Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • Received:2011-12-20 Revised:2012-04-12 Online:2012-10-14 Published:2012-10-14
  • Contact: Zhang Hong-qi, Doctor, Chief physician, Doctoral supervisor, Department of Spinal Surgery, Xiangya Spinal Surgery Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China zhq9996@163.com
  • About author:Wang Yu-xiang☆, Doctor, Department of Spinal Surgery, Xiangya Spinal Surgery Center, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China wangyuxiang628@hotmail.com

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Abstract:

BACKGROUND: There are many studies on how estrogen receptor (ER) α participates in bone metabolism at present. However, the studies of how ER β participates in bone metabolism are few.
OBJECTIVE: To construct a retroviral expression vector of RNA interference (RNAi) for human ER β and to mediate its expression in human MG63 osteoblast-like cells via retrovirus.
METHODS: Gene nucleotide sequence of ER β was retrieved from Genebank database. Three target small interfering RNA (siRNA) sequences were designed and converted into cDNA coding expression of small hairpin RNAs (shRNA) for ER β gene. The cDNA was synthesized and inserted into pRNAT-H1.4/Retro plasmid.The recombinant of shRNA eukaryotic expression vector-pRNΑT-H1.4/Retro-ERβ-shRNΑ was constructed and identified by restriction enzyme digestion and the sequence analysis. The recombinant vector was transfected into 293 cells by Lipofectamine 2000 and packaged as retrovirus. The blank and nonspecific shRNA of the packaged retrovirus served as controls. Human MG63 osteoblast-like cell strain was transfected.
RESULTS AND CONCLUSION:Three retroviral vectors-ERβ-shRNA (pRNΑT-H1.4/ Retro-ERβ-shRNΑ1, pRNΑT-H1.4/Retro-ERβ-shRNΑ2 and pRNΑT-H1.4/Retro-ERβ-shRNΑ3) were constructed. Enzyme digestion identification and sequence analysis had confirmed that the recombinant plasmid containing target sequence for ER β gene had been inserted into the site which was expected to meet the design requirements. The recombinant plasmid had been constructed successfully and was packaged as antivirus after transfected into 293 cells by Lipofectamine 2000. These findings suggest that retrovirus can transfect human MG63 osteoblast-like cell strain efficiently and stably. The transfecting rate was around 70%. Three kinds of ERβ-shRNA retrovirus: pRNΑT-H1.4/Retro-ERβ-shRNΑ1, pRNΑT-H1.4/Retro-ERβ-shRNΑ2 and pRNAT-H1.4/ Retro-ERβ-shRNΑ3 all can transfect human MG63 osteoblast-like cells efficiently and stably, as well as inhibit the expression of ERβ remarkably, and ERβ-shRNA3 is the most efficient shRNA sequence.

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