PMID: 22340474Feb 22, 2012Paper

Construction of a bicistronic recombinant adenoviral vector for human interleukin-10 and enhanced green fluorescent protein expression in bone marrow mesenchymal stem cells

Chinese Medical Journal
Jian-Qing LinYou-Guang Gao

Abstract

Human interleukin-10 (hIL-10) is a cytokine synthesis inhibitory factor, which is involved in various immune responses. The purpose of this study was to construct an adenoviral vector carrying the hIL-10 gene for expression of biologically active hIL-10 in rat bone marrow mesenchymal stem cells (rMSCs). A pSNAV2.0-hIL10 plasmid was used as a template to obtain a hIL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene. The pDC316-hIL-10-IRES-EGFP plasmid was linearized by PmeI digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax. Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange. After the number of virus particles and titer was determined, rMSCs were infected with the adenoviral vector. The infection rate was determined by fluorescence microscopy and flow cytometry, and hIL-10 protein expression in rMSCs was measured by Western blotting. The virus particle concentration, OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2 × 10(11) VP/m...Continue Reading

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