利用CRISPR/Cas9构建甲基丙二酸血症cblC型W203X突变小鼠模型

Construction of a mouse model of cblC type methylmalonic acidemia with W203X mutation based on the CRISPR/Cas9 technology

目的利用CRISPR/Cas9技术构建与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。方法通过BLAST比对人和小鼠cblC基因和蛋白序列的保守性,应用CRISPR/Cas9技术进行小鼠受精卵显微注射,获得杂合子F1代小鼠,F1代小鼠通过杂交获得W203X纯合突变型小鼠,并对纯合突变型、同窝杂合型及野生型3种类型小鼠进行血代谢产物丙酰肉碱检测。结果人和小鼠甲基丙二酸血症cblC型的致病基因MMACHC的核苷酸和氨基酸序列高度保守。通过CRISPR/Cas9技术成功获得W203X纯合突变型小鼠,该小鼠模型在生后24 h丙酰肉碱明显升高(P<0.001)。结论利用CRISPR/Cas9技术成功构建了与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。

Objective To construct a W203Xutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology.Methods At first,BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice,and then,the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice.Hybridization was performed for these mice to obtain homozygous W203X-mutant mice.The blood level of the metabolite propionyl carnitine(C3)was measured for homozygous mutant mice,heterozygous littermates,and wild-type mice.Results The gene and protein sequences of MMACHC,the pathogenic gene for cblC type methylmalonic acidemia,were highly conserved in humans and mice.The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology,and there was a significant increase in C3 in these mice at 24 hours after birth(P<0.001).Conclusions A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.