利用 CRISPR/ Cas9 技术构建 Bpi 基因敲除小鼠
作者:
作者单位:

1.济南大学/ 山东省医学科学院 医学与生命科学学院,济南 250200; 2.山东省实验动物中心,山东第一医科大学 (山东省医学科学院),济南 250002

作者简介:

通讯作者:

中图分类号:

R-33

基金项目:


Construction of Bpi gene-knockout mice using CRISPR / Cas9
Author:
Affiliation:

1.School of Medicine and Life Science, University of Jinan & Shandong Academy of Medical Science, Jinan 250200, China. 2. Shandong Laboratory Animal Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250002

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的 利用 CRISPR/ Cas9 基因编辑技术高效构建 Bpi 基因敲除的小鼠模型,并继续繁殖、鉴定及建立稳定遗传的 Bpi 基因敲除小鼠品系。 方法 针对 C57BL/ 6 小鼠 Bpi 基因的第 2~3 外显子两端设计敲除靶点,筛选高活性 gRNA(guide RNA)靶点,体外转录得到 sgRNA(small guide RNA)并与 Cas9 mRNA 一起显微注射到 C57BL/ 6 小鼠的受精卵内,经胚胎移植至代孕母鼠体内,获得 F0 代小鼠后,通过基因型鉴定和 DNA 测序获得基因突变的阳性子 代鼠。 结果 筛选到高活性 gRNA 靶点并成功获得体外转录产物 sgRNA;与 Cas9 mRNA 一起通过显微注射的方式获 得了 64 枚状态良好的受精卵并成功移植到 2 只代孕母鼠体内;获得了 22 只 F0 代小鼠,经 PCR 鉴定和 DNA 测序后选择一只单链缺失 708 bp 碱基的阳性小鼠进行扩繁,并在 F1、F2 代检测到该突变,且 F2 代成功繁育出纯合子小鼠。 结论 成功构建 Bpi 基因敲除小鼠模型,为进一步研究 Bpi 基因及其表达产物的生物学功能奠定了基础。

    Abstract:

    Objective To use CRISPR/ Cas9 gene editing technology to efficiently construct Bpi gene-knockout mouse models and continue to breed, identify and establish stable genetic Bpi gene-knockout mouse strains. Methods Knockout targets were designed at the two ends of exons 2-3 of the Bpi gene in C57BL/ 6 mice, and high activity guide RNA ( gRNA) targets were selected. The small guide RNA(sgRNA)was transcribed in vitro and microinjected into fertilized eggs of C57BL/ 6 mice together with Cas9 mRNA. F0 mice were obtained after embryo transfer into the surrogate mice. Positive mice with gene mutation was confirmed via genotype identification and DNA sequencing. Results Microinjection with Cas9 mRNA yielded highly active sgRNA and 64 fertilized eggs in good condition, which were successfully transplanted into 2 surrogate mice, yielding 22 F0 mice. After PCR identification and DNA sequencing, a mouse that was positive for a 708 bp single- strand deletion was selected to be propagated. The deletion was detected in the F1 and F2 generations, and homozygous mice were obtained in the F2 generation. Conclusions Bpi-knockout mouse models were successfully constructed, laying a foundation for further study of the biological functions of the Bpi gene and its expression products.

    参考文献
    相似文献
    引证文献
引用本文

张 雨,郭中坤,付 彬,雷 战,聂爱蕊,庄峰锋,王可洲.利用 CRISPR/ Cas9 技术构建 Bpi 基因敲除小鼠[J].中国比较医学杂志,2020,30(6):39~46.

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2019-11-28
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2020-07-23
  • 出版日期:
自2024年1期开始,杂志参考文献改为中英文对照,具体格式要求可置下载中心查看!
关闭