人乳头瘤病毒16 E6/7转基因小鼠模型的建立

目的 构建pCEP4-HPV16 E6/7真核表达质粒,通过显微注射法建立HPV16 E6/7转基因小鼠模型。方法 应用基因重组技术,构建pCEP4-HPV16 E6/7真核表达质粒,将经线性化的包含启动子、目的基因、PolyA尾的完整的真核表达DNA,通过将外源基因显微注射到供体小鼠受精卵的雄原核中,再植入到假孕受体鼠的输卵管中,获得129只F0代小鼠.常规法提取这些转基因小鼠的基因组DNA,进行PCR分析,得到5只原代转基因小鼠,再进行DNA印迹分析,其中4只小鼠的DNA出现与阳性质粒对照类似的特异条带,说明E6/7整合在这4只小鼠的基因组中。将Founder小鼠与正常FVB配种,后代经PCR检测,统计结果符合孟德尔遗传定律,证明外源基因稳定遗传给后代。在4只5月龄F₀代阳性转基因小鼠中有2只小鼠耳朵皮肤出现不典型增生改变。结果 成功构建了pCEP4-HPV16 E6/7真核表达质粒,用包含CMV启动子、HPV16 E6/7目的基因转基因的小鼠已出现皮肤组织不典型增生。结论 获得可繁殖传代的整合了HPV16 E6/7基因的转基因小鼠模型。

Establishment of human papillomavirus type 16 E6/7 transgenic mouse model

Objective To construct pCEP4-human papillomavirus type 16(HPV16)E6/7 gene eukaryotic expression plasmid,and to set up HPV16 E6/7 transgenic mouse model by microinjection. Methods Genetic recombination technique was used to construct eukaryotic expression plasmid pCEP4-HPV16 E6/7.The recombinant plasmid was microinjected into male pronuclei of donor mice,which were thentransplanted into fertilized ovum of pseudopregnant mice.Totally,129 founder mice were born. Of them,5 were confirmed by PCR to be transgenic mice.As Southern blot showed,4 of the 5 transgenic mice yielded specific fragments similar to the positive plasmid,which suggested that E6/7 gene was integrated into the genome of these 4 mice.The founder mice were mated with normal FVB mice,and pCEP4-E6/7 gene was detected by PCR in their descendants.The results confirmed that the exogenous gene had been stably inherited by their descendants.Epithelial dysplasia of skin tissue was observed in 2 of 4 5-months-old trans-genic mice.Results The eukaryotic expression plasmid pCEP4-HPV16 E6/7 was constructed successfully, and epithelial dysplasia of skin tissue was observed in its transgenic mice.Conclusion HPV16 E6/7 trans-genic mouse model,with the capability of propagating and passaging,is constructed successfully.