Construction of a mouse pDsRed2-N1-SDF-1alpha eukaryotic expression vector and its expression in mouse bone marrow mesenchymal stem cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector pDsRed2-N1-SDF-1alpha and observe its expression in the mouse bone marrow mesenchymal stem cells.</p><p><b>METHOD</b>SDF-1alpha gene sequence with XhoI, EcoRI restriction enzyme cutting site was amplified from the total RNA of mouse smooth muscle cells by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the eukaryotic expression vector pDsRed2-N1 encoding red fluorescent protein gene, and the insertion was verified by endonuclease digestion and DNA sequencing. Mouse bone marrow mesenchymal stem cells identified with immunofluorescence assay for vimentin expression were transfected with the constructed plasmid pDsRed2-N1-SDF-1alpha, and the expression of sdf-1alpha was detected using immunofluorescence assay.</p><p><b>RESULTS</b>The DNA fragment amplified by PCR from the total RNA was identical to SDF-1alpha from the gene library, and an identical DNA fragment was also amplified from the recombinants. Sequence analysis confirmed the successful insertion of SDF-1alpha into the pDsRed2-N1 vector and the eukaryotic expression vector pDsRed2-N1-SDF-1alpha was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-1alpha expression 24 h after transfection with the recombinant vector.</p><p><b>CONCLUSION</b>The pDsRed2-N1-SDF-1alpha eukaryotic expression vector constructed is capable of expression of SDF-1alpha fusion protein in the mouse bone marrow mesenchymal stem cells.</p>