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小鼠超高硫角蛋白基因启动子的克隆及其表达活性分析
引用本文:李昊,王春生,宁方勇,梁洋,朴善花,安铁洙.小鼠超高硫角蛋白基因启动子的克隆及其表达活性分析[J].中国实验动物学报,2010,18(6):471-474,I0006.
作者姓名:李昊  王春生  宁方勇  梁洋  朴善花  安铁洙
作者单位:[1]东北林业大学生命科学学院,哈尔滨150040 [2]东北农业大学动物科技学院,哈尔滨150030
基金项目:国家自然科学基金,东北林业大学引进人才科研启动基金
摘    要:目的筛选在小鼠毛囊中具有高表达活性的内源启动子,为建立小鼠被毛特异表达外源蛋白的转基因技术奠定基础。方法以小鼠基因组为模板,克隆得到超高硫角蛋白基因启动子超高硫角蛋白(UHS),将其分别与pβgal-Basic和pAcGFP1-N1载体连接,构建了重组真核表达载体。采用阳离子脂质体法转染胎鼠组织块,分析其表达活性。结果转染后48 h,在蓝光激发条件下可以检测到绿色荧光蛋白(GFP)在小鼠毛囊区高表达,转染96 h后,表达减弱;此外,转染后48 h,βgal染色结果显示在皮肤块的毛囊区存在蓝色点状区域。结论 UHS启动子在小鼠毛囊中具有表达特异性。

关 键 词:超高硫角蛋白  启动子  分子克隆  活性分析

Cloning and Activity of Mouse Ultra-High Sulfur Keratin Gene Promoter
LI Hao,WANG Chun-sheng,NING Fang-yong,Liang Yang,PIAO Shan-hua,AN Tie-zhu.Cloning and Activity of Mouse Ultra-High Sulfur Keratin Gene Promoter[J].Acta Laboratorium Animalis Scientia Sinica,2010,18(6):471-474,I0006.
Authors:LI Hao  WANG Chun-sheng  NING Fang-yong  Liang Yang  PIAO Shan-hua  AN Tie-zhu
Institution:1.College of Life Science,Northeast Forestry University,Harbin 150040,China;2.College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)
Abstract:Objective Keratin is the main structure albumen of the mammal wool,which is the most important part of the skin,hair and nail,etc.The aim of this study was to screen ultra-high sulfur keratin promoter in the hair follicles of mouse skin,and provide a basis for establishment of a transgenic technique for hair-specific expression of exogeneous protein.Methods Regarding the genomic DNA from the mouse as a template,the mouse ultra-high sulfur keratin promoter(UHS) was cloned using molecular biologic methods,identified after conjugation with pMD18 simple vector and then insert it into pAcGFP-N1 plasmid,which had been excised the CMV promoter.After identification by PCR,a recombinant eukaryotic expression vector was constructed.At the same time,the promoter was linked to pβgal-basic,and recombination plasmid U-GFP and U-β-gal were obtained,respectively.The mouse skin tissue pieces were transfected by cationic liposomes of this plasmids.Results GFP expression was detected at mouse hair follicle region under blue light ilumination at 48 h after transfection,and the expression was decreased at 96 h after transfection.Moreover,at 48 h after transfection,blue dots of βgal staining were seen at the mouse hair follicle region.Conclusion The UHS promoter is a tissue-specific promoter in the mouse hair follicles.
Keywords:Ultra-high sulfur keratin  Promoter  Molecular cloning  Activity analysis  Mouse
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