[1]李中泰,李彦锋,刘晋祎,等.鼠Fscb/Cabyr双基因靶向敲除动物模型的建立和鉴定[J].第三军医大学学报,2020,42(19):1898-1906.
 LI Zhongtai,LI Yanfeng,LIU Jinyi,et al.Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout[J].J Third Mil Med Univ,2020,42(19):1898-1906.
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鼠Fscb/Cabyr双基因靶向敲除动物模型的建立和鉴定(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第19期
页码:
1898-1906
栏目:
基础医学
出版日期:
2020-10-15

文章信息/Info

Title:
Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout
作者:
李中泰李彦锋刘晋祎 张勇张军毕罡
陆军军医大学(第三军医大学)大坪医院泌尿外科;陆军军医大学(第三军医大学)军事预防医学系毒理学研究所;中国人民解放军第990医院泌尿外科
Author(s):
LI Zhongtai LI Yanfeng1 LIU JinyiZHANG Yong ZHANG Jun BI Gang

Department of Urology, Daping Hospital, Army Medical University (Third Military Medical University), Chongqing, 400042; 2Institute of Toxicology, Faculty of Military Preventive Medicine, Army Medical University (Third Military Medical University), Chongqing, 400038; 3Department of Urology, No. 990 Hospital of PLA, Zhumadian, Henan Province, 463000, China

关键词:
基因敲除精子鞭毛 CRISPR/Cas9系统动物模型
Keywords:
gene knockout sperm flagella CRISPR / cas9 system animal model
分类号:
R-322; R321.1; R394-33
文献标志码:
A
摘要:

目的利用CRISPR/Cas9系统构建鼠Fscb/Cabyr双基因靶向敲除小鼠模型,为研究鼠FSCB和CABYR蛋白复合物在精子鞭毛运动和获能中作用奠定基础。方法 根据 Cabyr基因全长序列,设计CRISPR/Cas9作用靶点,合成4条单链向导RNA(single-guide RNA,sgRNA),克隆入pUC57-T7-gRNA表达载体,获得sgRNA表达质粒。sgRNA和Cas9 mRNA体外转录后,将Cas9 mRNA和sgRNA以适当比例混合并注入C57BL/6J小鼠受精卵中,获得Cabyr基因敲除的初建鼠(F0)。PCR鉴定后,选取两只嵌合体雌鼠与背景鼠回交繁育,获得靶基因敲除F1杂合鼠,将其与前期已经建立的Fscb杂合鼠交叉繁育,获得Fscb/Cabyr(--/--)双基因敲除纯合子小鼠。通过Western blot和免疫荧光检测双基因敲除后小鼠精子中FSCB/CABYR蛋白的表达情况。结果首先获得7只(3只雄鼠,4只雌鼠)删除Cabyr基因片段并产生移码的F1代杂合鼠,将F1代杂合雄鼠与雌鼠交配,获得Cabyr基因敲除纯合子小鼠。同时,以Fscb杂合小鼠与Cabyr杂合小鼠通过反复交叉繁育,最终获得Fscb/Cabyr(--/--)双基因敲除纯合子小鼠。Western blot检测证实双基因敲除纯合子雄鼠睾丸及精子内FSCB和CABYR 蛋白表达均消失,免疫荧光检测显示精子主段内无FSCB、CABYR蛋白荧光表达。结论应用CRISPR/Cas9 系统和杂合子交叉繁育成功获得Fscb/Cabyr(--/--)双基因敲除纯合子小鼠,为进一步研究FSCB和CABYR蛋白复合物的功能奠定了动物模型基础。

Abstract:

ObjectiveTo construct a mouse model of targeted knockout of Fscb/Cabyr dual genes by using the CRISPR/Cas9 system, in order to found a basis for the study of the role of FSCB and CABYR protein complexe in sperm flagellum movement and capacitation. MethodsThe targeting sites for CRISPR/Cas9 system were designed according to the full-length sequence of Cabyr gene. Two pairs of single-guide RNA (sgRNA) were chemically synthesized, and then inserted into the linearized plasmid pUC57-T7-gRNA to express sgRNA. The Cas9 RNA and sgRNA were transcribed in vitro by T7 RNA polymerase, and the obtained products were mixed in proper proportion and microinjected into the fertilized eggs of C57BL/6J mice to obtain the primary mouse (F0) with Cabyr gene knockout. After the identification of gene mutation by PCR, 2 chimeric females mice and background mice were selected for backcross breeding, and the Cabyr gene knockout F1 heterozygous mice were obtained. Then they were cross bred with the previously established Fscb heterozygous mice, and finally the Fscb/Cabyr (--/--) double gene knockout homozygous mice were obtained. The deletions of Fscb/Cabyr proteins in double gene knockout homozygous mice were detected by Western blotting and immunofluorescence assay. ResultsSeven positive F1 mice (3 males and 4 females) with deletion of the Cabyr gene fragment and frame shifting were obtained. The homozygous Cabyr knockout mice were obtained by mating the heterozygous male mice with the female mice. Fscb/Cabyr (--/--) double gene knockout homozygous mice were obtained by repeated cross breeding of Fscb and Cabyr heterozygous mice. Western blot analysis indicated that the expression of FSCB and CABYR protein disappeared in the testis and spermatozoa of the male mice. Immunofluorescence assay showed that there was no fluorescence expression of FSCB and CABYR protein in the principle piece of the sperm flagella. ConclusionFscb/Cabyr (--/--) double gene knockout homozygous mice are obtained by using the CRISPR/Cas9 system and cross breeding, which laid the foundation of animal model for further study on the function of FSCB and CABYR protein complex.

更新日期/Last Update: 2020-10-02