生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (4): 114-.doi: 10.3969/j.issn.2095-1736.2023.04.114

• 技术方法 • 上一篇    下一篇

TRPM2基因敲除小鼠的建立与鉴定

王存连， 李龙飞， 张瑞华， 徐明举， 徐　彤   

  1. 河北北方学院 动物科技学院， 张家口 075000
  • 出版日期:2023-08-18 发布日期:2023-08-10
  • 通讯作者: 徐彤，博士，教授，研究方向为动物流感病毒诱导哺乳动物肺损伤，E-mail:xutong1969@sohu.com
  • 作者简介:王存连，高级实验师，研究方向为流感病毒致肺损伤机理，E-mail:3349135107@qq.com
  • 基金资助:
    河北省第二期现代农业产业技术体系蛋肉鸡创新团队建设专项经费项目（HBCT2018150207）； 河北省自然科学基金资助项目（C2022405008, C2022405010）

Establishment and identification of TRPM2 gene knockout mice

WANG Cunlian, LI Longfei, ZHANG Ruihua, XU Mingju, XU Tong   

  1. College of Animal Science Technology, Hebei North University, Zhangjiakou 075000, China
  • Online:2023-08-18 Published:2023-08-10

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摘要： 利用CRISPR/Cas9技术构建瞬时受体电位M2（TRPM2）基因敲除的杂合子小鼠模型。参考Ensembl数据库中Trpm2-203的内含子2和内含子24序列选择sgRNA靶点并进行体外合成。通过体外转录方式获得Cas9 mRNA，将其与sgRNA显微注射至C57BL/6J小鼠的受精卵后，移植到假孕母鼠的输卵管内，获得F0代小鼠。阳性F0代小鼠分别与野生型C57BL/6J小鼠交配获得F1代。结果显示，经PCR产物测序共获得3只阳性F0代小鼠；阳性F0代小鼠分别与野生型C57BL/6J小鼠交配获得4个品系，共获得27只阳性F1代小鼠。通过实时荧光定量PCR和Western Blot方法验证，表明研究成功构建TRPM2基因敲除杂合子小鼠(TRPM2+/-)，为下一步开展TRPM2基因及其表达产物的生物功能研究提供良好的动物模型。

关键词: CRISPR/Cas9, TRPM2, 基因敲除, 小鼠

Abstract: The aim of this study was to obtain the transient receptor potential M2 (TRPM2) gene knockout heterozygous mouse model by using CRISPR/cas9 technology. Referring to intron 2 and intron 24 sequences of Trpm2-203 in Ensembl database, sgRNA targets were selected and synthesized in vitro. Cas9 mRNA was obtained by in vitro transcription. Cas9 mRNA and sgRNA were microinjected into the fertilized eggs of C57BL/6J mice and transplanted into the fallopian tube of pseudopregnant female mice to obtain F0mice. Positive F0generation mice were mated with wild-type C57BL/6J mice to obtain F1generation. Three positive F0generation mice were obtained by sequencing PCR products. Four strains were obtained by mating between positive F0generation mice and wild-type C57BL/6J mice with a total of 27 positive F1generation mice. Verified by real time fluorescent quantitative PCR and Western Blot, it was shown that the TRPM2 knockout heterozygous mice (TRPM2+/-) were successfully constructed in this study, which provides a good animal model for the further study of the biological function of TRPM2 gene and its expression products.

Key words: CRISPR/Cas9, TRPM2, gene knockout, mice

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