Mito-cpYFP cDNA转基因小鼠的建立及其自由基分布规律的初步探讨

目的:利用可实时动态测量超氧阴离子自由基的环状排列黄色荧光蛋白(circularly permuted yellow fluorescent protein,cpYFP)构建可实时动态监测活体动物体内自由基的转基因动物模型。方法:应用转基因技术将载有可定位于线粒体内的cpYFP(mitochondria-targeted cpYFP,mito-cpYFP)的载体pRP(Exp)-CAGGmitocpYFP注射到C57BL/6小鼠的受精卵中,采用PCR及RT-PCR的方法鉴定阳性小鼠,通过荧光观察自由基的分布并辅助鉴定阳性小鼠。结果:PCR、RT-PCR及荧光观察结果表明mito-cpYFP cDNA转基因小鼠构建成功。通过与野生型C57BL/6小鼠交配,得到F1、F2和F3代的新生幼鼠共计494只,其中,阳性幼鼠为255只,阳性率为51.6%。转基因阳性鼠体内出现了绿色荧光,其解剖伤口、肠道、脑部及口腔黏膜等荧光明显,而肾脏、肝脏及胸腺等荧光则较弱,耻骨联合部和膀胱出现绿色荧光,但约0.5 h后消失。结论:我们成功制备了可稳定表达并遗传mito-cpYFP的转基因小鼠品系。在活体动物体内,自由基在黏膜层含量较多。此模型可研究自由基在生物体内的含量及分布规律,为后续生物自由基作为信号分子在机体内传导途径的研究提供有效的工具。

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.