Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout

Zhongtai LI; Yanfeng LI; Jinyi LIU; Yong ZHANG; Jun ZHANG; Gang BI

doi:10.16016/j.1000-5404.202005029

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Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout

更新时间：2026-01-09

- Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout
- Journal of Third Military Medical University Vol. 42, Issue 19, Pages: 1898-1906(2020)
- 作者机构：
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81571491)
- DOI：10.16016/j.1000-5404.202005029
  CLC：
- Received：06 May 2020，
  
  Published：15 October 2020
- 稿件说明：
移动端阅览
Zhongtai LI, Yanfeng LI, Jinyi LIU, et al. Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout[J]. Journal of Third Military Medical University, 2020, 42(19): 1898-1906.
DOI：

Zhongtai LI, Yanfeng LI, Jinyi LIU, et al. Establishment and identification of a mouse model of Fscb/Cabyr double gene knockout[J]. Journal of Third Military Medical University, 2020, 42(19): 1898-1906. DOI： 10.16016/j.1000-5404.202005029.

摘要

Abstract

Objective

To 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.

Methods

The 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.

Results

Seven 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.

Conclusion

Fscb/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.

关键词

Keywords

references

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