小鼠精子蛋白基因fscb基因条件打靶载体的构建 - 第三军医大学学报.docVIP

鼠fscb基因靶向敲除载体的构建及动物模型的建立 周庭友，孙中义，张勇，张军，毕罡，刘旭东，李珂，周波，靳风烁，张克勤，李彦锋△ （第三军医大学大坪医院野战外科研究所泌尿外科，重庆 400042） 作者简介：周庭友（1984～），医师，硕士，主要从事男科学研究。△通讯作者，TelE-mail：lyf1000@ [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，合成三对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。将Cas9 mＲNA 和sgＲNA 以适当比例混合后注射入B6J小鼠受精卵中，获得基因敲除的初建鼠。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 成功构建了存在fscb基因突变的两种纯合子小鼠，小鼠睾丸内FSCB的蛋白表达消失。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。 [关键词] 基因敲除 精子蛋白 CRISPR/Cas9 动物模型 The construction of targeting vectors using CRISPR/Cas9 and the generation of fscb knockout mice Zhou Ting-You, Sun Zhong-Yi, Zhang Yong, Zhang Jun, Bi Gang, Liu Xu-Dong, Li Ke, Zhou Bo, Jin Feng-Shuo, Zhang Ke-Qin, Li Yan-Feng (Department of Urology，Research Institute of Surgery，Daping Hospital，Third Military Medical University，Chongqing 400042, China) Abstract] Objective To build the targeting vectors for the deletion of mice fscb gene by using the CRISPR/Cas9 system, and to generate fscb gene knockout mice for the studying of its function in the sperm activation and capcitation. Methods The sgRNA targeting sites were designed according to the full length sequence of fscb gene. Three sgRNAs 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 mices with targeted mutation．The genotype of homozygous were identified by PCR and DNA sequencing. Results Two lines of homozygous mice with fscb mutations were successful generated. The expression of FSCB protein in the mice testis and sperm were disappeared. Conclusions The fscb gene knockout model was successfully build by using the CRISPR/Cas9 system, which offered a tool for the studying of the function of FSCB protein. [Key words]