应用CRISPR/Cas9技术制备MrgD基因敲除小鼠模型

目的 利用CRISPR/Cas9基因编辑技术建立MrgD基因敲除的小鼠模型，为研究MrgD基因在糖、脂代谢中的作用提供实验工具。方法 根据MrgD基因第一外显子碱基序列，设计针对MrgD基因的sgRNA（single guide RNA），构建sgRNA表达质粒，利用T7RNA聚合酶体外转录sgRNA和Cas9后，将Cas9 mRNA和sgRNA对C57BL/6J小鼠的受精卵进行显微注射。使用基因测序检测MrgD基因碱基的突变情况。结果 获得了MrgD基因突变的F2代纯合子小鼠，即MrgD基因缺失173bp的小鼠。并且，初步研究显示MrgD基因纯合突变小鼠在普通饲料喂养下糖、脂代谢正常。结论 成功构建了MrgD基因敲除小鼠动物模型，为探讨MrgD基因在糖尿病发生发展中的作用提供研究平台。

Establishment of MrgD knockout mice model with CRISPR/Cas9 gene targeting technology

Objective To establish the MrgD gene knockout mice model by using CRISPR/Cas9 gene editing technique so as to provide the tool to study the regulating role of MrgD in glucose and lipid metabolism. Methods Single guide RNA (sgRNA) plasmids against the exon 1 of MrgD were designed and constructed. Then the sgRNA and Cas9 was transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA were mixed and microinjected into fertilized eggs of C57BL/6J mice. Gene sequencing was used to detect the mutations of MrgD. Results Finally the F2 generation of homozygous MrgD deficient mice (MrgD-173/-173) were gained. Preliminary studies showed that MrgD gene homozygous mutant mice may not have disorders of glucose and lipid metabolism. ConclusionA mouse model with MrgD deficiency has been successfully established which shall lay a foundation for future investigation of MrgD.