G蛋白偶联受体激酶2杂合子敲除小鼠构建及表型鉴定  

Generation of GRK2 heterozygous mouse using CRISPR/Cas9 and genotyping

作　　者：陶娟 周伟杰 邰宇[1] 郭派派 周正炜 王珍 汪庆童[1] 魏伟[1] Tao Juan;Zhou Weijie;Tai Yu(Institute of Clinical Pharmacology,Anhui Medical Unwersity,Key Laboratory of Anti-inflammatory and Immune Medicine Ministry of Education,Anhui Collaborative Innovation Center cf Anti-inflammatory and Immune Medicine,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所抗炎免疫药物教育部重点实验室安徽抗炎免疫药物协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2020年第7期1035-1041,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81202541、81973332、81973314);安徽省自然科学基金杰出青年基金(编号:1808085J28);安徽高校自然科学研究重点项目(编号:KJ2017A176);安徽省高校优秀青年人才支持计划项目(编号:gxyqZD2017025);安徽省留学回国人员创新创业扶持计划(2017);安徽医科大学青年拔尖人才支持计划(2013);第三批安徽医科大学校级中青年学术技术带头人基金。

摘　　要：目的应用CRISPR/Cas9技术构建G蛋白偶联受体激酶2(GRK2)杂合敲除(GRK2^+/-)小鼠,用于疾病病理机制及药物靶点研究。方法构建靶向敲除GRK2基因的载体,在体外将先导RNA和Cas9 mRNA显微注射到C57BL/6小鼠的受精卵中,在小鼠GRK2基因的第3个外显子上造成基因突变,通过PCR及基因测序测定F0代基因型,成功获得13bp和19bp敲除两种GRK2^+/-小鼠。因GRK2纯合子敲除胚胎期死亡,将19bp敲除F0代小鼠与C57BL/6小鼠回交,获得稳定的19bp敲除F1代GRK2^+/-小鼠用于扩繁及实验。结果 GRK2^+/-小鼠基因型稳定,体重较同龄C57BL/6小鼠低,Western blot检测证实GRK2^+/-小鼠心脏、脾脏、胸腺、肝脏、巨噬细胞及肾脏组织中GRK2蛋白的表达均显著降低。结论该研究利用CRISPR/Cas9技术成功建立了GRK2^+/-小鼠模型,为进一步研究GRK2在多种脏器、细胞发育中的作用,疾病发病机制及药物作用靶点提供了重要的动物模型。Objective To generate a G protein-coupled receptor kinase 2(GRK2) heterozygous(GRK2^+/-) mice by CRISPR/Cas9 for investigating the pathogenic role and therapeutic implication of GRK2.Methods GRK2 targeting vector was constructed and the mixture of single guide RNA and Case9 mRNA was microinjected into zygotes of female C57 BL/6 mouse to generate gene mutation in Exon3. We got both 13 bp-deletion and 19 bp-deletion GRK2^+/- miceaccording to genotyping performed through PCR and gene sequencing. Since GRK2 homozygous are embryonic lethality,we cross-bred 19 bp-deletion F0 GRK2^+/- mice with C57 BL/6 to obtain stable 19 bp-deletion F1 generation of GRK2^+/- mice for propagation and the following experiments. Results The genotype of GRK2^+/- mouse was steady with significant lower body weight compared with C57 BL/6 mouse at the same age. Western blot results showed that almost half of the GRK2 protein expression was reduced in immune organs like spleen and thymus as well as other tissues. Conclusion This GRK2 targeted genetically engineered mouse model will provide a promising approach for understanding the role of GRK2 in the development of diverse organs and cells as well as its pathogenic role and therapeutic implications in chronic diseases.

关 键 词：G蛋白偶联受体激酶2 CRISPR/Cas9 基因工程小鼠 基因型鉴定 

分 类 号：R394-33[医药卫生—医学遗传学]