鼠fscb基因靶向敲除载体的构建及动物模型的建立

发布时间：2018-01-07 06:09

本文关键词：鼠fscb基因靶向敲除载体的构建及动物模型的建立　出处：《第三军医大学学报》2015年15期 　论文类型：期刊论文

【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。
[Abstract]:Objective to construct a mouse FSCB gene targeting vector by CRISPR/Cas9 system, for the study of mouse FSCB protein in the role of the establishment of the FSCB gene knock-out animal model of sperm flagellar motility and capacitation. Methods according to the sequence of the full-length FSCB gene design targets of CRISPR/Cas9, the synthesis of 3 of single wizard RNA (single-guide RNA, sgRNA), and cloned into PU57-T7-GDNA vector by PCR, sequencing, sgRNA expression plasmid. The use of T7 RNA polymerase in vitro transcription of sgRNA and Cas9 RNA. Cas9mRNA and sgRNA mixed in a proper ratio after B6J injected into mouse fertilized eggs, FSCB gene knockout mice (F0). First built by PCR to identify the gene mutation, selection of target gene knockout mice were sequenced and further analysis to determine the mutation. The expression vector sgRNA were constructed successfully and in vitro transcription, will have a direct injection of sgRNA and Cas9 in rat RNA activity The fertilized eggs, obtained 39 F0 positive mice, a total of 3 clear and delete gene frameshift in male rats and female rats received 5 backcross breeding wild F1 generation positive mice (2 males, 3 females), F1 heterozygous male and female mice mated to obtain homozygous mice. Through the identification, established FSCB gene two genotypes of knockout mice, and cultured breeding of.Western blot analysis confirmed that two mice homozygous male mice testis and sperm in the expression of FSCB protein disappeared. Conclusion through CRISPR/Cas9 system targeting FSCB gene knockout and homozygous mice, providing knockout in addition to a mouse model for further analysis of the function of FSCB protein.

【作者单位】：第三军医大学大坪医院野战外科研究所泌尿外科;
【基金】：国家自然科学基金面上项目(81170617)~~
【分类号】：R-332
【正文快照】： 已有研究发现严重弱精症或精子完全无运动的患者存在精子纤维鞘发育不良[1],精子形成过程中睾丸特异性基因的适时有序表达,并驱动精子各细胞器的有序形成包括精子鞭毛骨架结构和相关蛋白的有序装配整合是保证精子发挥正常功能的根本物质基础[2]。纤维鞘CABYR结合蛋白(fibrous

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