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.
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.
Hu
Weihua
W
Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. huweihua1980@sina.com
Guo
Fengjing
F
Li
Feng
F
Huang
Hui
H
Zhang
Weikai
W
Chen
Anmin
A
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology*; D002356:Cartilage / Q000166:cytology*; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D051381:Rats; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D055758:SOX9 Transcription Factor / Q000096:biosynthesis / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology*; D014162:Transfection
D011994:Recombinant Proteins; D055758:SOX9 Transcription Factor; C496366:Fgfr3 protein, mouse; D051498:Receptor, Fibroblast Growth Factor, Type 3