Hr突变体转基因小鼠的构建和表型分析

The Establishment and Phenotypic Analysis of Hr Transgenic Mice

无毛基因(Hr)定位于8p12,在染色体上跨越14 kb,包含19个外显子。无毛基因的自发突变能引起人和动物毛发脱落及相关毛发疾病的产生。为深入研究Hr基因的功能,本文利用Gateway技术构建Hr表达载体,在该基因的3 427位引入点突变(G→A),通过显微注射建立转基因小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6小鼠回交后互交数代建系。观察转基因小鼠毛发生长发育规律。结果表明,成功构建了pRP(Exp)-EF1A>m Hairless mutant>IRES/EGFP真核表达载体,通过与野生型小鼠杂交获得阳性子代,进行同窝交配,第2代小鼠出生后14 d开始脱发,30 d左右脱落的毛发重新长出。取部分皮肤组织做石蜡切片,皮肤组织学观察发现,脱毛期无毛小鼠毛囊瓦解,真皮内形成大小不等的包囊,毛发重新生长时,真皮内见大量新生的毛囊。蛋白印迹实验表明,转基因小鼠脱发时HR蛋白表达量明显高于同龄阴性小鼠。本文成功建立稳定遗传的Hr突变的转基因小鼠品系,推测无毛基因突变引发转基因小鼠的脱发,为研究Hr基因的功能提供了良好的动物模型。

Human hairless gene（ Hr） contains 19 exons that lies at the position of chromosome 8p12.Mutations in the Hr gene in both mice and human have been implicated in the development of atrichia.Full length c DNAs of hairless gene（ Hr） were cloned and a point mutation at 3 427（ G → A） was introduced to study the function of Hr. The expression plasmid constructed by gateway clone technology was microinjected into the fertilized eggs from C57 BL/6 mice. The Hr transgenic mice were generated by microinjection method and the genotype of gene was identified by PCR. The heterozygote mice were gradually eliminated by intercrossing the descendants. The phenotype and skin histology of the transgenic mice were studied and compared to haired littermates. The HR protein expression in the transgenic mice was assessed by Western blot analysis. The results showed that recombinant plasmid pRP（ Exp）-EF1 A 〉m Hairless mutant 〉 IRES/EGFP was constructed and the independent homozygous transgenic strains were established. It was shown that the transgenic mice grew a first coat of hair and appeared normal until 14 days after birth when loss of hair began. The transgenic mice developed alopecia except for the hair on head after 2 weeks. Histologic appearance of skin in the transgenic mice exhibited characteristic features,including loss of hair shaft and multiple dermal cysts. Interestingly,new hair regrew typically about 30 days and the hair coat developed normally for its life. The amount of HR protein expression determined by Western blot analysis was elevated in the transgenic mice compared to haired littermates. The Hr mutant transgenic mice explain at least in part that the hair loss could be induced by mutation of Hr. These findings provide further understanding for the mechanism of disorder of hair growth and development.