Construction Of Inducible Lentiviral Expression Vector Containing HBMP-2 Gene And Its Expression In Human Mensechymal Stem Cell

Objective To construct inducible lentiviral vector containing human bone morphogenetic protein-2(hBMP-2) gene and harvest lentiviral-hBMP-2 viruses from contransfected 293FT,which infects human mensechymal stem cells with hBMP-2gene expression under doxcycline induction and provides a basis for further study of transfecting human cord blood mensechymal stem cells to bone necrosis.Methods hBMP-2 gene carrying attB recombinant sites was amplified by PCR from pcDNA-hBMP-2 plasmid. Using the Gateway recombination cloning technology, hBMP-2 gene was subcloned into pDown by BP reaction. After confirmation of a correct construction by sequencing, pLV/EXPN2-Neo-TRE-hBMP-2 was obtained by LP reaction among pDown-hBMP-2,pUp-TRE and pLV/Des2-Neo. The recombinant vectors was contransfected into 293FT respectively through lipofectamine, and the lentiviral-hBMP-2 viruses were harvested from 293FT.Viruses were used to infect human cord mensechymal stem cells, which can express target gene hBMP-2 under the induction of doxcycline.Results Inducable lentiviral vector containing hBMP-2 gene was successfully detected through enzyme digestion and sequencing.pLV/EXPN2-Neo-TRE-hBMP-2 was contransfected into 293FT. Lentiviral-hBMP-2 viruses were harvested successfully and were highly efficient to infect human cord mensechymal stem cells which express hBMP-2 under doxcycline induction.Conclusion The recombinant inducable lentiviral vectors containing hBMP-2 gene has been successfully constructed and lentiviral-hBMP-2 viruses were harvested,which is viable to obtain inducible human cord mensechymal stem cells with hBMP-2gene expression under doxcycline induction and provides a basis for further study of transfecting human cord blood mensechymal stem cells to bone necrosis.