心肌锚定重复序列蛋白基因心脏特异敲除小鼠模型构建与鉴定

Construction and identification of mice model with cardiac disruption of cardiac ankyrin repeat protein

目的构建心肌锚定重复序列蛋白(CARP)基因心脏特异敲除小鼠模型,为研究CARP基因与心脏疾病的关系奠定基础。方法构建CARP基因条件打靶载体pL253-CARP-LoxP电转入小鼠胚胎干细胞(ES)细胞,G418和GANC药物筛选阳性细胞克隆,Southern blot确定CARP基因条件打靶载体转染成功。胚胎注射ES细胞入小鼠囊胚,获得嵌合体小鼠与心脏特异启动子Cre小鼠(α-MHC-Cre)杂交。结果得到6只嵌合体小鼠,与α-MHC-Cre小鼠杂交获得CARP基因心脏特异敲除小鼠(CARP-KO),半定量RT-PCR和Western blot确定CARP基因在CARP-KO小鼠心脏缺失。该小鼠早期胚胎发育和个体生长、心脏发育正常,但心肌肥厚标志基因β-MHC和ANF显著升高(分别为2.25±0.04和1.45±0.03)(P<0.05)。结论成功构建CARP基因心脏特异敲除小鼠,CARP基因缺失对小鼠胚胎发育、个体生长和心脏发育没有显著影响。

Objective To establish a mouse model with cardiac disruption of cardiac ankyrin repeat protein（CARP） and to develope reliable tools for studying the function of CARP gene in heart related diseases.MethodsConstruct the conditional target plasmid of pL253-CARP-LoxP and transfect to 129 embryonic stem cells.Positive clones selected by G418 and GANC were checked by Southern blot to confirm conditional disruption of CARP gene,and then injected to the blastula.Chimeras were got and crossed with α-MHC-Cre transgenic mice,which expressed recombinase specifically in heart.Results Six chimeras were developed,and cardiac disruption of cardiac ankyrin repeat protein mice（CARP-KO） were obtained after crossing with α-MHC-Cre mice.CARP gene disruption was confirmed in the heart of CARP-KO mice by semi-quantitative RT-PCR and Western blot.These mice displayed normal in embryonic development,body growth,and heart development.However the expression of hypertrophic genes of β-MHC and ANF increased significantly in CARP-KO mice（2.25±0.04 and 1.45±0.03,respectively）（P0.05）.Conclusions The mouse model with specific disruption of CARP gene was successfully developed.The cardiac disruption of CARP gene does not the hamper development and growth of mice significantly,neither the heart development.