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Construction of hTAZ Expressing Lentiviral Vector and theEffect of hTAZ on Differentiation of MC3T3-E1 Preosteoblast

Author: ZhangZuo
Tutor: ShiXueTao；YinZhanHai
School: Fourth Military Medical University
Course: Biomedical Engineering
Keywords: osteoporosis cell differentiation lentivirus TAZ
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 15
Quote: 0
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Abstract


Objective: To construct hTAZ expressing lentiviral vector pLenti6/V5-hTAZand study the regulatory effect of hTAZ on the differentiation of MC3T3-E1preosteoblast. Methods: LR reaction was performed between pENTR221-hTAZ plasmid which contain hTAZ cDNA and pLenti6/V5-DEST plasmid.hTAZ cDNA was cloned into lentiviral vector pLenti6/V5-DEST. The newrecombinant was identified by restriction enzyme digestion and sequencing.The overexpression of hTAZ in cells was validated by Western blot. Thepseudoviral particles containing the expression construct of hTAZ weregenerated by lentiviral packaging system in 293FT cells and employed toinfect MC3T3-E1 cells. Clonal MC3T3-E1 cells were selected by blasticidinadded. MC3T3-E1 wild type（MC3T3-E1）and hTAZ overexpressing（MC3T3-E1/TAZ）were induced by osteogenic medium and adipogenicmedium to observe effect of hTAZ on differentiation. Osteogenicdifferentiation was confirmed by von Kossa staining and alizarin red Sstaining while adipogenic differentiation by oil red O staining respectively. Results: Agarose electrophoresis and sequencing identified that hTAZ cDNAwas successfully cloned into lentiviral vector pLenti6/V5-DEST. Therecombinant plasmid could be expressed correctly. The pseudoviral particlescontaining hTAZ were constructed. MC3T3-E1/TAZ cell line was establishedafter pseudovirus infection and blasticidin selection. The von Kossa stainingand alizarin red staining results showed that mineralization of hTAZoverexpressing MC3T3-E1/TAZ was higher than that of MC3T3-E1 wild typecell line，while adipogenic differentiation of MC3T3-E1/TAZ was inhibited.Conclusion: The present experiments demonstrate successful cloning ofhuman hTAZ cDNA into the lentiviral vector pLenti6/V5-DEST and itsexpression in MC3T3-E1 cells. Overexpression of hTAZ can promoteosteogenic differentiation while inhibit adipogenic differentiation of MC3T3-E1 cell line.

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