Construction of Sox9 gene eukaryotic expression vector and its inductive effects on directed differentiation of bone marrow stromal cells into precartilaginous stem cells in rats |
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Authors: | Weihua Hu Fengjing Guo Feng Li Hui Huang Weikai Zhang Anmin Chen |
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Institution: | Department of Orthopedics, Tongji Hospital,Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,China |
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Abstract: | Summary Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order
to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by
Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2
expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal
cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry
was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive
endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were
successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and
FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded
that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal
cells differentiated into the precartilaginous stem cells.
This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30650006). |
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Keywords: | precartilaginons stem cells Sox9 bone marrow-derived stromal cells transfection |
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