Construction of lentiviral vector containing SOX9 gene and its expression in bone marrow mesenchymal stem cells

Abstract

To construct the lentiviral vector containing SOX9 gene and to detect its expression in MSCs derived from rabbit bone marrow. Human sox-9 gene coding region fragment was obtained by RT-PCR (reverse transcription-polymerase chain reaction) and then cloned into the plasmid of Pwpxl-MOD2 to form Pwpxl-MOD2/SOX9. Pwpxl-MOD2/SOX9, pRsv-REV, pMDlg-Prre and pMD2G were co-transfected into 293T cells to obtain recombinant virus containing SOX9 gene. Meanwhile, Pwpxl-MOD2, pRsv-REV, pMDlg-pRRE and pMD2G were transfected into another group of 293T cells as a control group packing into blank lentiviral vector. Then the packed lentiviral vector was transfected into MSCs which derived from rabbit bone marrow. The expression of SOX9 was detected by both RT-PCR and Western blot. Identification and proliferation of MSCs was determined by MTT after transfection. The sequencing and restriction analysis showed that SOX9 gene fragment was correctly connected and cloned into the plasmid Pwpxl-MOD in lentiviral vectors. After transfection, the expression of SOX9 gene in MSCs was confirmed by RT-PCR and Western blot. MTT showed the growth of MSCs had no significant effect after transfection with lentiviral vector. Lentiviral vector carrying SOX9 gene has been successfully constructed. There is a stable expression in transfected MSCs. Thus it will facilitate the exploratory development of gene and biological therapy for intervertebral disc degeneration.