Expression of human bone morphogenetic protein-2 gene regulated by Tet-On system

Abstract

Objective To construct an effective Tet-On expression system for transferring human bone morphogenetic protein- 2 (hBMP - 2 ) gene into bone marrow mesenchymal progenitor cells (BMSCs), and to determine its expression with the presence of low dose doxycycline (DOX) induction. Methods The hBMP-2 cDNA was subcloned into the pTRE-Shuttle2 vector by molecular cloning and cloned into adenovirus to construct the recombinant Adeno-X-hBMP-2 vector. The recombinant linear DNA fragments Adeno-X-hBMP-2 was transfected into the human embryo kidney 293 (HEK293) cells by lipofectamine mediated gene transferring. At the same time aclenovirus vector containing transcriptional activator BD Adeno-X Tet-On virus Stock was transfected into the HEK293 cells by the same method to get amplification. BMSCs were co-infected with the virus of Adeno-X-hBMP-2 and the Adeno-X Tet-On. The expression of hBMP-2 mRNA was identified by RT- PCR induced by DOX. The dose- effect relationship between DOX and hBMP-2 was examined by ELISA at different DOX concentrations. Results Tet-On regulation system encoding hBMP-2 gene was constructed successfully at the level of BMSCs. The obvious expression of hBMP-2 mRNA was detected with RT-PCR. The amount of hBMP-2 protein was (165.677±0.008) ng/L with 0.01 mg/L DOX as found by ELISA. DOX inducing concentration and expressing amount of hBMP- 2 detected by ELISA demonstrated positive dose- dependent correlation while the level of DOX was higher than 0.01 mg/L. Conclusions An effective Tet- On expression system for transferring hBMP- 2 gene into BMSCs is constructed successfully. After co-infected by this system, BMSCs can significantly express hBMP-2 induced by low-dose DOX. The production of hBMP-2 is dose-dependent with the DOX level above 0.01 mg/L. Key words: Bone morphogenetic proteins; Transfection; Mesenchymal stem cells; Tet-On; Bone defect