利用CRISPR/Cas9技术构建ALK7^(LoxP/LoxP)小鼠品系  

Establishment of ALK7^(LoxP/LoxP) mouse by CRISPR/Cas9

作　　者：赵强[1] 刘灯泉 李毅辉 戴红艳 唐梦熊[3] 管军[1] ZHAO Qiang;LIU Dengquan;LI Yihui;DAI Hongyan;TANG Mengxiong;GUAN Jun(Department of Cardiology,Affiliated Qingdao Municipal Hospital of Qingdao University,Qingdao,Shandong 260000;ICU,Qilu Hospital of Shandong University,Jinan,Shandong 250012;Department of Emergency,Qilu Hospital of Shandong University,Jinan,Shandong 250012,China)

机构地区：[1]青岛大学附属青岛市市立医院心内科,山东省青岛市266000 [2]山东大学齐鲁医院重症医学科,山东省济南市250012 [3]山东大学齐鲁医院急诊科,山东省济南市250012

出　　处：《中国动脉硬化杂志》2019年第1期69-74,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81670411)

摘　　要：目的利用CRISPR/Cas9技术将LoxP序列靶向导入小鼠活化素受体样激酶7(ALK7)基因,构建ALK7LoxP/LoxP小鼠,与组织特异性Cre小鼠杂交,获得时空特异性ALK7基因敲除小鼠,为研究ALK7在特定时间特定组织中的功能奠定基础。方法利用CRISPR/Cas9技术编辑小鼠ALK7基因:设计合成识别ALK7基因外显子4-6上下游非编码序列的sg RNA。设计并合成LoxP-ALK7-LoxP打靶载体,测序正确后,显微注射法将体外合成的sg RNA、Cas9 m RNA和打靶载体注射到小鼠受精卵,移植受精卵至假孕小鼠输卵管代孕。获得仔鼠后通过PCR、Southern blot鉴定子代小鼠基因型,实时荧光定量PCR、Western blot检测ALK7转录表达水平。结果获得了含有目的基因的打靶阳性小鼠,并且插入的LoxP序列不影响ALK7的转录表达水平。结论利用CRISPR/Cas9技术成功将LoxP序列靶向引入小鼠ALK7基因,成功构建ALK7LoxP/LoxP小鼠品系,为进一步构建组织特异性ALK7基因敲除小鼠模型奠定了基础。Aim To generate mutant mouse with ALK7 gene inserted by LoxP sequences by CRISPR/Cas9,to further develop temperospatial specific ALK7 deleted mouse models via cross bred with the tissue-specific Cre mouse,providing a basis for functional study of ALK7 in special time and tissue.Methods The CRISPR/Cas9 technology was used to edit ALK7.Two sg RNAs were designed to direct Cas9 endonuclease cleavage in intron 3-4 and intron 6-7.The targeting vector with LoxP-ALK7-LoxP sequence was designed.sg RNA,Cas9 m RNA and targeting vector were co-injected into zygotes,which were transferred to pseudopregnant mice.The pups were genotyped by PCR and Southern blott.Real-time PCR and Western blot were performed for analysis of the expression of ALK7.Results The ALK7LoxP/LoxP mouse was generated by CRISPR/Cas9,and did not show influence on the expression of ALK7.Conclusion The CRISPR/Cas9 technology can successfully generate the ALK7LoxP/LoxPmouse by inserting LoxP sequence into ALK7 gene,which is a basis for the creation of tissue-specific ALK7 deleted mouse models.

关 键 词：CRISPR/Cas9 活化素受体样激酶7基因 LoxP序列 组织特异性敲除 

分 类 号：Q81[生物学—生物工程] R5[医药卫生—内科学]