大理大学学报 ›› 2022, Vol. 7 ›› Issue (2): 33-38.

• 基础医学 • 上一篇    下一篇

应用CRISPR/Cas9技术构建fto基因敲除小鼠模型

华 卉12,夏 静12,贾慧婕12,葛禹辰12,汪小波12*   

  1. 1.大理大学基础医学院,云南大理 6710002.云南省高校细胞生物学重点实验室,云南大理 671000

  • 收稿日期:2021-03-17 修回日期:2021-09-14 出版日期:2022-02-15 发布日期:2022-03-29
  • 通讯作者: 汪小波,副研究员,博士,E-mail: wxb4320062@163.com。
  • 作者简介:华卉,硕士研究生,主要从事细胞衰老研究。
  • 基金资助:
    国家自然科学基金项目(81860158);云南省地方本科高校(部分)基础研究联合专项资金项目(2017FH001-074

Construction of fto Knockout Mouse Model with CRISPR/Cas9 Gene Targeting Technology

Hua Hui12 Xia Jing12 Jia Huijie12 Ge Yuchen12 Wang Xiaobo12*   

  1. 1. Pre-clinical College Dali University Dali Yunnan 671000 China 2. Yunnan Key Laboratory of Cell Biology in Universities Dali Yunnan 671000 China

  • Received:2021-03-17 Revised:2021-09-14 Online:2022-02-15 Published:2022-03-29

摘要:  目的:通过CRISPR/Cas9基因编辑技术构建fto基因敲除小鼠模型,为FTO功能研究提供材料。方法:体外将Cas9 mRNA和靶向fto的向导RNAguide RNAgRNA)显微注射到小鼠受精卵中,通过聚合酶链式反应、扩增及产物测序、蛋白质印迹法对小鼠fto基因的敲除进行鉴定及验证。结果:成功将具有活性的gRNA载体和Cas9 mRNA注射入受精卵中,获得3F0代阳性初建鼠。经同种小鼠杂交配种,最终得到fto基因敲除的纯合子小鼠。结论:通过CRISPR/Cas9技术成功制备了fto基因敲除小鼠模型,为进一步研究fto基因功能奠定了基础。

关键词: font-family:", ">"> , ">ftofont-family:SimSun, ">基因;肥胖;基因敲除小鼠;font-family:SimSun, ">CRISPR/Cas9

Abstract: Objective This study aims to provides materials for FTO function research by constructing fto knockout mouse model with CRISPR/Cas9 gene targeting technology. Methods Cas9 mRNA and guide RNAgRNA targeting fto were microinjected into mouse fertilized eggs in vitro and the knockout of mouse fto gene was identified and verified by ploymerase chain reactionPCR), PCR product sequencing and Western-Blotting. Results The active gRNA vector and Cas9 mRNA were injected into the fertilized egg successfully in vitro and three positive F0 mice were obtained at last. The homozygous mice with fto gene knockout were finally obtained by hybridization and mating with the same mice. Conclusion The fto gene knockout mouse model through CRISPR/Cas9 technology was successfully prepared which laid a foundation for further research on the function of fto gene.

Key words: font-family:", ">"> , ">ftofont-family:Times New Roman, ">;font-family:Times New Roman, "> obesityfont-family:Times New Roman, ">;font-family:Times New Roman, "> knockout mousefont-family:Times New Roman, ">;font-family:Times New Roman, "> CRISPR/Cas9

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