Construction of pLenti6/V5-DEST-TAZ vector and its effect on differentiation of MC3T3-E1 preosteoblasts

Objective: To construct TAZ expressing lentivirus vector pLenti6/V5-DEST-TAZ and study the regulatory effect of TAZ on the differentiation of MC3T3-E1 preosteoblasts. Methods: LR reaction was performed to clone the pENTR (tm) 221-TAZ plasmid containing TAZ cDNA into pLenti6/V5-DEST plasmid. The new recombinant plasmid was identified by restriction enzyme. The overexpression of TAZ in cells was validated by Western blotting. The pseudoviral particles containing the expressed construct were generated by lentiviral packaging system in 293FT cells, which were used to infect MC3T3-E1 to select monoclonal cells by using Blastcidin added. MC3T3-E1 wild type cell line and TAZ overexpressing cell lines were induced towards osteoblasts with condition medium. Differentiation and mineralization of two cell lines were verified by assay of Von Kossa staining and Alizarin red staining, respectively. Results: Agarose electrophoresis and sequencing examination showed that TAZ cDNA was cloned into lentiviral vector pLenti6/V5-DEST. The recombinant plasmid could be expressed correctly. The pseudoviral particles with TAZ were obtained and the MC3T3-E1 cell lines were infected successfully. The Von Kossa staining and Alizarin red staining results showed that the mineralization of TAZ overexpressing cell lines was higher than that of MC3T3-E1 wild type cell line. Conclusion: The pLenti6/V5-DEST-TAZ is constructed successfully, and it can express in the cells correctly. The human TAZ cDNA is cloned into the lentiviral vector pLenti6/V5-DEST. The results indicate that the overexpression of TAZ can promote the differentiation of MC3T3-E1 cell line into osteoblasts.