上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

肾小管上皮细胞C/EBPα条件性敲除小鼠的构建及鉴定

应霁， 刘剑， 张沥文， 仲芳， 周芳芳， 王伟铭   

  1. 上海交通大学 医学院附属瑞金医院肾脏科, 上海 200025
  • 出版日期:2016-10-28 发布日期:2016-11-29
  • 通讯作者: 王伟铭， 电子信箱: wweiming01@126.com。
  • 作者简介:应霁（1990—）, 女， 硕士生； 电子信箱: yingji1990@sina.cn。
  • 基金资助:

    国家自然科学基金（81270782，30771000）；国家重点基础研究发展计划（“973”计划）（2012CB517701, 2012CB517604）；科技部十二五科技攻关项目（2011BAI10B00, 2011BAI10B06）；上海市卫计委重中之重学科建设项目；卫生行业科研专项项目（201002010）；比较性研究，上海市科研计划项目（15140902800）

Generation and identification of renal tubular epithelial cell C/EBPα conditional knockout mice

YING Ji, LIU Jian, ZHANG Li-wen, ZHONG Fang, ZHOU Fang-fang, WANG Wei-ming   

  1. Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2016-10-28 Published:2016-11-29
  • Supported by:

    National Natural Science Foundation of China, 81270782， 30771000; National Basic Research Program of China, 2012CB517701, 2012CB517604; the National Science & Technology Pillar Program during the Twelfth Fiveyear Plan Period, 2011BAI10B00, 2011BAI10B06; Key Discipline Construction Project of Shanghai Municipal Commission of Health and Family Planning; Health Industry Specific Research Project, 201002010; Comparative Research, Scientific Research Project of Shanghai, 15140902800

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摘要：

目的·构建肾小管上皮细胞C/EBPα条件性敲除小鼠，并进行表型鉴定，为进一步研究C/EBPα在肾间质纤维化中的作用奠定基础。方法·利用C/EBPαflox/flox和pepck-Cre+转基因小鼠进行饲养和杂交；将繁殖产生的第1代小鼠再次进行交配，得到第2代小鼠。通过PCR鉴定出基因型为C/EBPαflox/flox.pepck-Cre+的小鼠（C/EBPα-/-小鼠）。采用RT-qPCR及免疫组织化学法分别检测肾组织中C/EBPα mRNA和蛋白表达水平。应用H-E染色、Masson染色观察6~8周龄C/EBPα-/-小鼠及野生型（WT）小鼠肾脏组织形态学改变。结果·2种转基因小鼠繁殖产生的第2代小鼠基因型符合孟德尔遗传定律，交配后获得C/EBPα-/-小鼠。与WT小鼠相比，C/EBPα-/-小鼠肾脏中C/EBPα mRNA及蛋白表达水平下降，光学显微镜下肾脏组织形态无明显改变。结论·应用Cre-loxp系统可以成功地敲除小鼠肾小管上皮细胞中C/EBPα基因，且在基础情况下C/EBPα-/-小鼠肾脏组织无明显形态学改变。

关键词: Cre-loxp系统；C/EBP&alpha, ；条件性敲除；肾小管上皮细胞

Abstract:

Objective·To generate renal tubular epithelial cell C/EBPα conditional knockout  mice, perform phenotypic identification, and lay a foundation for further study on the role of C/EBPα in renal interstitial fibrosis.Methods·C/EBPαflox/flox and pepck-Cre+ transgenic mice were bred and hybridized.The first generation offspring were mated to reproduce the second generation. Mice with C/EBPαflox/flox.pepck-Cre+ genotype (C/EBPα-/- mice) were identified using PCR. The mRNA and protein expressions of C/EBPα in renal tissues were measured using RT-qPCR and immunohistochemistry staining. The morphologic changes in renal tissues of C/EBPα-/- mice and wide type (WT) mice aged 6-8 weeks were observed using H-E staining and Masson staining.Results·The genotype of the second generation offspring of two types of transgenic mice accorded with the Mendelian laws and C/EBPα-/- mice were generated. C/EBPα-/- mice had lower mRNA and protein expressions of C/EBPα in renal tissues and no significant renal morphologic changes compared with WT mice.Conclusion·Cre-loxp system can successfully knock out the C/EBPα gene in mouse renal tubular epithelial cells. C/EBPα-/- mice have no significant renal morphologic changes in baseline condition.

Key words: Cre-loxp system； C/EBPα； conditional knockout, renal tubular epithelial cells