Transfection of recombinant adenoviral vector with co-expressing keratinocyte growth factor and enhanced green fluorescent protein to murine bone marrow mesenchymal stem cells / 中国实验血液学杂志

ABSTRACT

To construct the adenoviral vector with co-expressing keratinocyte growth factor (KGF) and enhanced green fluorescent protein (EGFP) for transfection into the mesenchymal stem cells (MSC), the target gene KGF was cloned into the shuttle plasmid with the report gene EGFP, then the recombinant shuttle plasmid was transformed into DH5a bacteria to recombine with backbone vector pAdxsi. Next, the plasmid pAd-EGFP-mKGF was amplified in H293 cells and the viral titer was determined. The MSC were separated and enriched by using bone marrow adherent culture and identified in vitro to observe the efficiency of transfection. The results indicated that the recombinant shuttle plasmid pShuttle-EGFP-mKGF digested with restriction endonucleases was confirmed by two products which length was about 0.6 kb and 5.1 kb, respectively; the recombinant plasmid pAdxsi-EGFP-mKGF digested with restriction endonucleases was confirmed by 7 products; recombinant adenoviral vector Ad-EGFP-mKGF was amplified to titer of 1.6 × 10(10) pfu/ml. At 10 h after transfecting MSC began to express fluorescence at 6 to 8 days later, the fluorescence reached to the peak with infection rate of 92.3, at 28 days the expression of fluorescence was still observed. It is concluded that the recombinant adenoviral vector Ad-EGFP-mKGF is successfully constructed and can transfect MSC effectively and safely.