利用CRISPR/ Cas9 基因编辑技术建立FcγR 基因大片段敲除小鼠模型
作者:
作者单位:

(1. 中国食品药品检定研究院实验动物资源研究所,国家啮齿类实验动物种子中心,北京 102629;2. 中国食品药品检定研究院国家药物安全评价监测中心,北京 100176)

作者简介:

通讯作者:

中图分类号:

Q95-33

基金项目:


Establishment of a large fragment FcγR gene knockout mouse model using CRISPR / Cas9 genome editing technique
Author:
Affiliation:

(1. National Center for Laboratory Rodents, National Institutes for Food and Drug Control, Beijing 102629,China.2. National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing 100176)

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 使用CRISPR/ Cas9 基因编辑技术敲除长度约为90 kb 的小鼠FcγR2b, FcγR3, FcγR4 基因簇,为构建FcγR 基因人源化小鼠奠定基础?方法 使用在线预测软件在FcγR2b, FcγR3 外显子区设计sgRNA,在每一位点挑选脱靶效应较低的五个候选sgRNA?通过CRISPR/ Cas9 活性检测试剂盒检测sgRNA 在体外的活性?选取活性较高的sgRNA 体外转录,与Cas9 mRNA 一并注射受精卵?通过PCR 检测及测序,得到1 只敲除片段为89 711 bp 的基因修饰小鼠,且同时敲除FcγR2b 基因5’端,FcγR3 基因3’端及FcγR4 基因?而且还利用软件预测了8 个脱靶可能性最高的位点,并对首建鼠基因组的上述8 个脱靶位点全部测序确认?结果 结果显示未在预测脱靶位点附近发现小片段插入或缺失?结论 建立了利用CRISPR/ Cas9 基因编辑技术敲除基因组超大片段的技术,该技术结合BAC 转基因技术,将为建立含有复杂基因族的人源化小鼠提供新的途径?

    Abstract:

    Objective The mouse FcγR2b, FcγR3, and FcγR4 gene clusters of about 90 kb in length wereknocked out by CRISPR/ Cas9 genome editing technique to lay the foundation for constructing an FcγR gene humanizedmouse. Methods Single guide RNAs (sgRNAs) were designed using online software to target exons of FcγR2b andFcγR3. Five candidate sgRNAs with lower off-target effects were selected for each target site. The cleavage activity ofsgRNAs was tested in vitro by the CRISPR/ Cas9 activity assay kit. The sgRNA with high cleavage activity was selected forin vitro transcription and microinjected with Cas9 mRNA into zygotes. Results A genetic modified mouse with 89,711 bpknockout fragment was confirmed by PCR detection and sequencing. The 5′ end of the FcγR2b gene, the 3′ end of theFcγR3 gene and FcγR4 gene were knocked out at the same time. Moreover, 8 sites with the highest probability of off-targetwere predicted by software, and all 8 off-target sites of the founder mouse genome were sequenced and confirmed. No smallfragment insertion or deletion was found in any predicted off-target sites. Conclusions We established a large fragmentknockout technique using CRISPR/ Cas9 genome editing technique, which may provide a new method to establish humanized mouse models by combining with BAC transgenic strategies.

    参考文献
    相似文献
    引证文献
引用本文

吴曦,霍桂桃,刘甦苏,谷文达,曹愿,柳全明,吕建军,范昌发.利用CRISPR/ Cas9 基因编辑技术建立FcγR 基因大片段敲除小鼠模型[J].中国实验动物学报,2019,27(5):583~591.

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:2019-03-01
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2019-11-04
  • 出版日期:
征稿啦 |“科技伦理前沿谈”征文通知
关闭