基于CRISPR/Cas9构建小鼠UOX基因敲除模型

Construction of Mouse UOX Gene Knockout Model Based on CRISPR/Cas9

目的通过CRISPR/Cas9获得UOX基因敲除的小鼠纯合品系,为建立高尿酸血小鼠动物模型奠定基础。方法根据小鼠UOX基因第三外显子前后两个位点设计双sgRNA,通过PCR、体外转录和纯化获得sgRNA和Cas9 mRNA。将sgRNA和Cas9 mRNA显微注射进小鼠原核胚后,体外培养至二细胞胚阶段,进行胚胎移植至代孕母鼠。F0代小鼠出生后,提取其DNA进行电泳分析和测序分析。F0代交配繁殖至F2代获得UOX缺失的小鼠纯合品系。结果显微注射sgRNA和Cas mRNA至原核胚,成功获得50枚囊胚。移植后生育17只幼鼠。其中,有8只小鼠UOX基因缺失突变,突变率为47.06%。成功构建了UOX缺失的小鼠纯合品系。结论利用CRISPR/Cas9,成功获得UOX缺失的小鼠胚胎和纯合品系,为CRISPR/Cas9获得高尿酸血的小鼠动物模型奠定基础。

Objective To obtain homozygous strains of UOX knockout mice by CRISPR/Cas9,and to lay the foundation for establishing an animal model of hyperuricemia mice.Methods Double sgRNA was designed based on two sites before and after the third exon of mouse UOX gene,and sgRNA and Cas9 mRNA were obtained by PCR,in vitro transcription and purification.After microinjection of sgRNA and Cas9 mRNA into mouse prokaryotic embryos,they were cultured in vitro to the two-cell embryo stage,and embryos were transferred to surrogate mother rats.After F0 mice were born,their DNA was extracted for electrophoretic analysis and sequencing analysis.FOX generations were bred to F2 generations to obtain UOX-deficient mouse homozygous lines.Results Microinjection of sgRNA and Cas mRNA into prokaryotic embryos successfully obtained 50 blastocysts.After transplantation,17 young rats were born.Among them,8 mice had UOX gene deletion mutations,and the mutation rate was 47.06%.A homozygous mouse line lacking UOX was successfully constructed.Conclusion UCRISPR-deficient mouse embryos and homozygous strains were successfully obtained using CRISPR/Cas9,laying a foundation for CRISPR/Cas9 to obtain hyperuricemia mouse animal models.