利用CRISPR/Cas9技术构建Bpi基因敲除小鼠  

Construction of Bpi gene-knockout mice using CRISPR/Cas9

作　　者：张雨 郭中坤 付彬[2] 雷战 聂爱蕊[1] 庄峰锋 王可洲 ZHANG Yu;GUO Zhongkun;FU Bin;LEI Zhan;NIE Airui;ZHUANG Fengfeng;WANG Kezhou(School of Medicine and Life Science,University of Jinan&Shandong Academy of Medical Science,Jinan 250200,China;Shandong Laboratory Animal Center,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250002)

机构地区：[1]济南大学/山东省医学科学院医学与生命科学学院,济南250200 [2]山东省实验动物中心,山东第一医科大学(山东省医学科学院),济南250002

出　　处：《中国比较医学杂志》2020年第6期39-46,共8页Chinese Journal of Comparative Medicine

基　　金：山东省医学科学院医药卫生科技创新工程、国家重点研发计划(2017YFD050160202);山东省自然科学基金(ZR2015YL036);山东省自然科学基金(ZR2016CP18)。

摘　　要：目的利用CRISPR/Cas9基因编辑技术高效构建Bpi基因敲除的小鼠模型,并继续繁殖、鉴定及建立稳定遗传的Bpi基因敲除小鼠品系。方法针对C57BL/6小鼠Bpi基因的第2~3外显子两端设计敲除靶点,筛选高活性gRNA(guide RNA)靶点,体外转录得到sgRNA(small guide RNA)并与Cas9 mRNA一起显微注射到C57BL/6小鼠的受精卵内,经胚胎移植至代孕母鼠体内,获得F0代小鼠后,通过基因型鉴定和DNA测序获得基因突变的阳性子代鼠。结果筛选到高活性gRNA靶点并成功获得体外转录产物sgRNA;与Cas9 mRNA一起通过显微注射的方式获得了64枚状态良好的受精卵并成功移植到2只代孕母鼠体内;获得了22只F0代小鼠,经PCR鉴定和DNA测序后选择一只单链缺失708 bp碱基的阳性小鼠进行扩繁,并在F1、F2代检测到该突变,且F2代成功繁育出纯合子小鼠。结论成功构建Bpi基因敲除小鼠模型,为进一步研究Bpi基因及其表达产物的生物学功能奠定了基础。Objective To use CRISPR/Cas9 gene editing technology to efficiently construct Bpi gene-knockout mouse models and continue to breed,identify and establish stable genetic Bpi gene-knockout mouse strains.Methods Knockout targets were designed at the two ends of exons 2-3 of the Bpi gene in C57BL/6 mice,and high activity guide RNA(gRNA)targets were selected.The small guide RNA(sgRNA)was transcribed in vitro and microinjected into fertilized eggs of C57BL/6 mice together with Cas9 mRNA.F0 mice were obtained after embryo transfer into the surrogate mice.Positive mice with gene mutation was confirmed via genotype identification and DNA sequencing.Results Microinjection with Cas9 mRNA yielded highly active sgRNA and 64 fertilized eggs in good condition,which were successfully transplanted into 2 surrogate mice,yielding 22 F0 mice.After PCR identification and DNA sequencing,a mouse that was positive for a 708 bp singlestrand deletion was selected to be propagated.The deletion was detected in the F1 and F2 generations,and homozygous mice were obtained in the F2 generation.Conclusions Bpi-knockout mouse models were successfully constructed,laying a foundation for further study of the biological functions of the Bpi gene and its expression products.

关 键 词：BPI 基因敲除小鼠 CRISPR/Cas9技术 基因型鉴定 

分 类 号：R-33[医药卫生]