鼠fscb基因靶向敲除载体的构建及动物模型的建立  

Construction of targeting vector and preparation of fibrous sheath CABYR-binding protein knockout mice using CRISPR / Cas9 system

作　　者：周庭友 孙中义[1] 张勇[1] 张军[1] 毕罡[1] 刘旭东[1] 李珂[1] 周波[1] 靳风烁[1] 张克勤[1] 李彦锋[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所泌尿外科,重庆400042

出　　处：《第三军医大学学报》2015年第15期1527-1533,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81170617)~~

摘　　要：目的利用CRISPR/Cas9系统构建鼠fscb基因打靶载体,为研究鼠FSCB蛋白在精子鞭毛运动和获能中作用建立fscb基因敲除动物模型。方法根据fscb基因全长序列,设计CRISPR/Cas9作用靶点,合成3对单链向导RNA(single-guide RNA,sgRNA),并克隆进PU57-T7-GDNA载体,通过PCR测序鉴定,获得sgRNA表达质粒。利用T7 RNA聚合酶体外转录sgRNA和Cas9 RNA。将Cas9mRNA和sgRNA以适当比例混合后注射入B6J小鼠受精卵中,获得fscb基因敲除的初建鼠(F0)。通过PCR对基因突变情况进行鉴定,选取靶基因敲除的小鼠进行传代并进一步测序分析确定突变情况。结果成功构建表达sgRNA的载体并进行体外转录,将有活性的sgRNA和Cas9 RNA直接注射入鼠受精卵,获得39只F0代阳性小鼠,选取3只明确删除目的基因片段并产生移码的雄鼠与野生雌鼠回交繁育得到5只F1代阳性小鼠(2只雄鼠,3只雌鼠),将F1代杂合子雄鼠与雌鼠交配,获得纯合子小鼠。通过鉴定,建立了两种不同基因型的fscb基因敲除鼠系,并传代繁育。Western blot分析证实两种鼠系纯合子雄鼠睾丸及精子内的FSCB蛋白表达消失。结论通过CRISPR/Cas9系统靶向敲除fscb基因并获得纯合子小鼠,为进一步分析FSCB蛋白的功能提供了基因敲除小鼠模型。Objective To build the targeting vectors to delete the gene of mouse fibrous sheath CABYR-binding protein （fscb） by using the CRISPR/Cas9 system, and to establish fscb gene knockout mice to study its function in the sperm activation and capacitation. Methods The targeting sites were designed according to the full-length sequence offscb gene, and 3 pairs of single-guide RNA （sgRNA） were chemically synthesized, and inserted into the linearized plasmid pUC57-T7-GDNA. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA mixtures were microinjected into fertilized eggs to generate mice with targeted mutation. The genotypes of homozygous were identified by PCR, DNA sequencing and Western blotting. Results The vector expressing sgRNA was successfully built, and the sgRNA and Cas9 mRNA were transcribted in vitro. The active sgRNA and Cas9 mRNA were then injected into the fertilized eggs of mice, and 39 positive mice as the F0 generation were generated. Three select male mice with clear deletion of the ficb gene fragment and with obvious frame shifting F1 mice （2 males and 3 females） were achieved. The were bred with wild-type female mice, and 5 positive heterozygous males mated with females to generate the 172 homozygous mice. Two different genotypes of homozygous were generated. Western blot analysis confirmed that the expression of FSCB protein in the testis and sperm of the homozygous male mice were disappeared. Conclusion The ficb gene knockout model is successfully established by using the CRISPR/Cas9 system, which offer a tool for the function of FSCB protein.

关 键 词：基因敲除 精子蛋白 CRISPR/Cas9 动物模型 

分 类 号：R332[医药卫生—人体生理学] R394.33[医药卫生—基础医学]