Establishment and identification of mito-cpYFP cDNA transgenic mice and preliminary studies on distribution of free radicals

AIM:To establish a transgenic animal model for real-time and dynamic detection of free radicals in vivo by expressing circularly permuted yellow fluorescent protein (cpYFP), which acts as a superoxide sensor in the living cells. METHODS:The plasmid of pRP(Exp)-CAGG>mito-cpYFP containing mitochondria-targeted cpYFP (mito-cpYFP) gene was microinjected into fertilized eggs from C57BL/6 mice. The breeding mice was identified by the methods of PCR, RT-PCR and fluorescence observation. RESULTS:The results of PCR, RT-PCR and fluorescence observation indicated that mito-cpYFP cDNA transgenic mouse model was established successfully. By mating with wild-type C57BL/6 mice, 3 generations of transgenic mice were obtained in a total number of 494. The number of cpYFP positive mice was 255, and the positive rate was 51.6%. Green fluorescence was observed in the cpYFP positive mice. The fluorescence in the wounds, intestines, brains and oral mucosa was obviously stronger than that in the kidney, liver and thymus. The fluorescence occurred in symphysis pubis and bladders, but disappeared in half an hour. CONCLUSION:A transgenic mouse strain that stably expresses and inherits mito-cpYFP was successfully constructed. Free radicals are mainly distributed in the mucosal layer in vivo according to fluorescence observation of mito-cpYFP cDNA transgenic mice. This model might be applied to study the content and distribution of superoxygen anion free radical in vivo. It might provide an effective approach for subsequent studies on free radical signaling pathways.