Construction of pECFP-C1- CIRP and its effect on cold-induced apoptosis in mouse NIH/3T3 cells
Cold-inducible RNA-binding protein (CIRP) is suggested to be involved in protecting cells from the damage caused by cold stress. In this study, the recombinant plasmid, pECFP-C1-CIRP, was constructed and transfected into NIH/3T3 cells by lipofectamineTM 2000. The fluorescence microscopy, real-time polymerase chain reaction (RT-PCR) and Western blot detection were done for transient and stable expression assays. CIRP gene was successfully subcloned into eukaryotic expression vector of pECFP-C1, confirmed by restrictive enzyme digestion analysis and DNA sequencing. Fluorescent microscopy study showed that green fluorescence was generated from NIH/3T3 cell. The results of RT-PCR indicated that the CIRP mRNA was expressed in NIH/3T3 cells, while the results of Western blot detection further indicated that the CIRP protein was overexpressed in NIH/3T3 cells. Stable expression in cells transfected with pECFP-C1-CIRP was achieved by selection in a G418-containing medium for 2 weeks. We performed experiments of cold-induced apoptosis under cold treatment of 4, 14 and 24°C. The results of the cell apoptosis detected by fluorescence microscopy indicated that the apoptosis level was reduced in pECFP-C1-CIRP stably expressed group due to overexpression of CIRP. This study suggested that CIRP played a role in protecting cells from cold-induced apoptosis.
Key words: Cold inducible RNA-binding protein, NIH/3T3 cell, transfection, cold-induced apoptosis.