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

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Author:
QIAN Dong-yang(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
BAI Bo(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
LIU Qi(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
YAN Guang-bin(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
WU Mei-xiang(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
WU Jing-ming(Department of Orthopaedics, First Affiliated Hospital, Gnangzhou Medical College, Guangzhou 510120, China)
Journal Title:
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
Issue:
Volume 15, Issue 04, 2009
DOI:
10.3760/cma.j.issn.1674-1927.2009.04.017
Key Word:
Bone morphogenetic proteins;Transfection;Mesenchymal stem cells;Tet-On;Bone defect

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.

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