【作者】 方亮；

【导师】 金伯泉； 缪军；

【作者基本信息】 第四军医大学 ， 免疫学， 2006， 硕士

【摘要】 目的：建立四环素调控的mPTA1／CD226转基因小鼠，对转基因小鼠胸腺细胞的发育进行研究。 方法：①使用pcDNA3-mPTA1和pBI-5载体构建用于四环素调控转基因小鼠制备的pBI-5-mPTA1载体。②采用显微注射的方法将pBI-5-mPTA1载体片段导入B6D1F1小鼠受精卵，对新生小鼠耳成纤维细胞进行体外培养，用含rtTA的pUHD17.1质粒转染细胞，选出荧光素酶表达活性依赖Dox的小鼠。③将阳性小鼠与正常C57BL／6小鼠交配，分别使用mPTA1和荧光素酶(luciferase)引物对子代小鼠的基因组DNA进行PCR检测和鉴定，选取双阳性的Ptet-mPTA1转基因小鼠的F1代，将其继续与C57BL／6小鼠交配，不断得到子代小鼠，直至F4代以后。④将阳性的Ptet-mPTA1转基因小鼠与携带有tTA的EμSR-tTA转基因小鼠杂交，得到子代小鼠，同时使用mPTA1和tTA引物对其基因组DNA进行PCR检测，选取mPTA1和tTA双阳性的小鼠。⑤使用荧光素酶检测试剂盒对杂交的阳性小鼠的不同组织，如胸腺、脾脏、心脏、肾脏等进行更多还原

【Abstract】 Objective: To establish the conditional tetracycline-regulated transgenic mice overexpressing mouse platelet and T cell activation antigen 1 （mPTA1） , and study the development of thymocytes in the transgenic mice.Method: The mPTAl expression vector was constructed by recombination of pcDNA3-mPTA1 and pBI-5, and the transgenic mouse lines were generated by microinjection using standard techniques. Positive animals were selected based on analysis of DNA sequence from mouse tails by PCR. The controlled regulation of the integrated expression unit was tested by transfection of the primary mouse ear fibroblasts obtained from DNA positive founders with the rtTA plasmid pUHD-17.1. Founder animals that showed DOX dependent expression of luciferase were selected and interbred with C57BL/6 mice to establish F1 of P_tetmPTA1 transgenic mice with defined genetic background.Double-transgenic animals containing the luciferase/mPTA1 transcription and expression tTA in thymocytes were obtained by crossing individuals of更多还原