Construction of lentivirus vector carrying CDX2 and its expression in bone mesenchymal stem cells

Objective To construct CDX2 gene in rat bone mesenchymal stem cells, exploring a mean of gene therapy for rat ulcerative colitis models. Methods Primers of CDX2 were designed according to the gene information from GenBank. Gene fragments of CDX2 were amplifed by polymerase chain reaction (PCR); The gene fragment and linear plasmid vector were connected by In-Fusion technology and transformed into competent cells. The positive clones of lentiviral expression vector were obtained after screening and followed by sequencing. The lentiviral vector was used to transfect 293T cells and package virus, and then the virus titers were determined. BMSCs were isolated and cultured in vitro. The immunophenotyple of BMSCs was detected by flow cytometry. The lentivirus vector containing CDX2 gene was transfected into rat BMSCs. The CDX2 mRNA and protein expression level were detected by RT-PCR and Western Blot. Results lentiviral vector encoding Ubi-CDX2-MCS-3FLAG-CMV-EGFP GV365 was successfully constructed, and confirmed by PCR, DNA sequencing，and western blot， and its titer reached 2×10⁸TU/ml.BMSCs were isolated and cultured successfully. FACS showed CD29, CD73, CD90 and CD105 were expressed on the cell sufaces, except for CD34 and CD45, ftting the phenotype of BMSCs.RT-PCR and Western blot detected that CDX2 gene was transfected into rat BMSCs and stably expressed, all of which showed the CDX2-BMSCs were constructed successfully. Conclusion BMSCs that carries and stably expresses gene CDX2 has been produced, which provides a new tool for experimental research of gene therapy in ulcerative colitis models.