摘要
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F_0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。
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 then transplanted into fertilized ovum ofpseudopregnant 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 HPV 16 E6/7 transgenic mouse model, with the capability of propagating and passaging, is constructed successfully.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2007年第5期286-288,共3页
Chinese Journal of Dermatology
基金
江苏省自然科学基金(BK2004057)
江苏省重点学科基金(135-3)
