人突变型p53和EB病毒LMP1转基因小鼠的建立

目的：建立含突变型p53和EB病毒LMP1基因的双转基因小鼠模型，为研究突变型p53和EB病毒LMP1基因在鼻咽癌前病变中的交互作用提供有力的工具．方法：通过分子克隆技术构建含突变型p53基因和EB病毒LMP1基因的真核表达载体pLMP1-p53mt；采用显微注射法将线性化的表达载体注射至小鼠受精卵的雄性原核中，然后将注射存活的受精卵植入假孕母鼠的输卵管；取其产3wk龄子代鼠进行PCR初选，再通过Southern杂交进一步确证；利用HE染色法观察4mo龄转基因小鼠不同组织病理变化．结果：将转基因载体进行了707枚小鼠受精卵的显微注射，然后植入30只假孕母鼠的输卵管中，其中26只母鼠怀孕，移植成功率为86．67％；共出生子代鼠179只；PCR检测显示179只子代小鼠中有9只整合了p53mt和LMP1基因，转基因阳性小鼠比例为5．03％（9／179），Southem杂交结果进一步证实了9只基因组整合了外源基因的首建者小鼠；随机抽取其中的1只转基因阳性小鼠鼻腔黏膜、鼻咽、食管表现炎症，鼻咽黏膜上皮灶性增生．结论：成功建立整合人突变型p53和LMP1的转基因小鼠，且表现鼻咽黏膜上皮灶性增生，可为鼻咽癌前病变机制的研究提供手段．

Construction of transgenic mice containing human mutant p53 and EB virus latent membrane protein 1

AIM: To establish bi-transgenic mice models containing mutation p53 gene and Epstein-Barr (EB) virus latent membrane protein 1 gene (LMP1) and to provide reliable tools for studying the reciprocity of mutant p53 with LMP1 in the tumorogenesis process of nasopharyngeal precancerosis. METHODS: Molecular cloning techniques were used to construct the recombinant plasmid pLMP1-p53mt containing mutant p53 gene and EB virus LMP1 gene. Linear recombinant plasmid pLMP1-p53mt was transfected into mouse androgenesis of germ cells by microinjection and then survival germ cells treated were planted into fallopian tubes of artificial pregnant female mice. Three-week filial generation mice were screened by PCR and further identified by southern blot, and the histopathological characteristics of different tissues in 4-month transgenic mice were observed by H-E staining. RESULTS: Seven hundred and seven mouse zygotes were treated with transgenic vectors DNA by microinjection and then transplanted into 30 artificial pregnant female mice. Twenty-six of these mice were gravid. The transplantation rate was 86.67%. A total of 179 F0 mice were obtained, of which 9 were carried p53mt gene and LMP1 gene screened by PCR. The positive rate of transgenic mice was 5.03% (9/179). The 9 positive F0 mice carrying foreign genes were further confirmed by southern blot. The rhinal mucosa, nasopharyngeal tissues and oesophagus of one founder had inflammation and its nasopharynx mucosa had focal hyperplasia. CONCLUSION: Transgenic mouse models integrating human mutant p53 gene and LMP1 and with focal hyperplasia of nasopharynx mucosa have been established by microinjection, which provides an effective model in studying the mechanism of nasopharyngeal precancerous lesions.