磷酸二酯酶4D纯合子敲除小鼠的构建  

Construction of phosphodiesterase 4D homozygous knockout mice

作　　者：朱振铎 苏甜甜 程慧娟 江春如 方茹红 管秋韵 何凤 葛明丽 魏伟[1] 汪庆童[1] Zhu Zhenduo;Su Tiantian;Cheng Huijuan;Jiang Chunru;Fang Ruhong;Guan Qiuyun;He Feng;Ge Mingli;Wei Wei;Wang Qingtong(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Education,Rheumatoid Arthritis Research Center,Anhui Medical University,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,安徽医科大学类风湿关节炎研究中心,合肥230032

出　　处：《安徽医科大学学报》2023年第2期208-213,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金资助项目(编号:81973314、81973332);安徽省杰出青年科学基金(编号:1808085J28);安徽高校协同创新项目(编号:GXXT-2020-066);2021年安徽省高校学科(专业)拔尖人才学术资助项目(编号:gxbjZD2021047)。

摘　　要：目的应用CRISPR/Cas9技术构建磷酸二酯酶4D(PDE4D)纯合子敲除小鼠,为深入探究PDE4D基因的功能及作用机制提供基础。方法针对PDE4D基因外显子4,5号构建载体显微注射到C57BL/6J小鼠受精卵中,经过母本繁育和子代交配获得PDE4D^(-/-)小鼠,通过PCR产物测序及基因型鉴定技术测定小鼠基因型。使用超声成像系统和HE染色检测小鼠主要器官形态和功能的变化,通过Westernblot实验验证PDE4D蛋白在小鼠体内的表达情况。结果PDE4D^(-/-)小鼠基因型稳定遗传,小鼠体型较小,体内主要器官无明显形态及组织学改变。PDE4D杂合或纯合敲除小鼠主要组织中PDE4D表达减少或基本不表达,敲除效果较好。结论本课题利用CRISPR/Cas9技术成功建立了PDE4D^(-/-)小鼠,未发现明显生理异常,可用于以PDE4D为靶点的疾病发病机制及药物研究。Objective The CRISPR/Cas9 technology was applied to construct PDE4D homozygous knockout mice to provide a basis for in-depth investigation of PDE4D gene function and mechanism of action.Methods A vector was constructed for PDE4D gene exon 4,5 microinjected into fertilized eggs of C57BL/6J mice,and PDE4D^(-/-)mice were obtained after maternal breeding and offspring mating,and the mice genotypes were determined by PCR product sequencing and genotype identification techniques.Changes in morphology and function of the major organs of the mice were detected using an ultrasound imaging system and H&E staining,and the expression of PDE4D protein in the mice was verified by Western blot assay.Results The PDE4D^(-/-)mouse genotype was stably inherited,the mice were small,and there were no obvious morphological and histological changes in the major organs in vivo.The PDE4D expression was reduced or largely absent in the major tissues of PDE4D heterozygous or pure knockout mice,and the knockout effect was better.Conclusion PDE4D^(-/-)mice were successfully established using CRISPR/Cas9 technology,and no significant physiological abnormalities were found,which could be used for disease pathogenesis and drug research using PDE4D as the target.

关 键 词：磷酸二酯酶4D CRISPR/Cas9 基因敲除 基因型鉴定 

分 类 号：R394-33[医药卫生—医学遗传学]