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目的建立心脏过表达人源PRKAG2(G100S)的转基因小鼠模型,为进一步研究该人源基因点突变对小鼠心脏发育、形态和功能维持的作用奠定基础。方法克隆人源基因PRKAG2并构建点突变质粒,将人源PRKAG2(G100S)插入α肌球蛋白重链(α-MHC)启动子下游,构建转基因表达载体。选用C57BL/6J小鼠为遗传背景,通过显微注射法建立人源PRKAG2(G100S)转基因小鼠模型,利用特异引物PCR法鉴定转基因小鼠的基因型。采用实时荧光定量PCR(qPCR)和蛋白质印迹法检测人源PRKAG2(G100S)的表达。结果经过回交繁育后建立了2个品系的心脏特异表达人源PRKAG2(G100S)的转基因小鼠品系F2代,并通过qPCR、蛋白质印迹法检测,确认了转基因小鼠心脏组织中人源PRKAG2(G100S)在mRNA和蛋白水平存在过表达,且该突变能在转基因小鼠中稳定传代。结论本研究成功建立了心脏特异表达人源PRKAG2(G100S)转基因小鼠模型,人源PRKAG2(G100S)基因在心脏组织的过度表达在小鼠心脏发育和功能维持中的作用需要进一步深入研究与探讨。
OBJECTIVE: To establish a transgenic mouse model of human heart PRKAG2 (G100S) overexpression, which lays the foundation for further study on the role of human point mutation in mouse heart development, morphology and function maintenance. Methods The human PRKAG2 gene was cloned and inserted into the plasmid pGEM-1. The human PRKAG2 (G100S) was inserted into the downstream of α-MHC promoter to construct the transgene expression vector. The C57BL / 6J mice were selected as the genetic background. The human PRKAG2 (G100S) transgenic mouse model was established by microinjection and the genotypes of the transgenic mice were identified by PCR. Human PRKAG2 (G100S) expression was detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting. Results F2 generation of transgenic mouse strains with heart-specific expression of human PRKAG2 (G100S) was established by backcrossed breeding. F2 generation of the transgenic mouse strains with heart-specific expression of human PRKAG2 (G100S) were confirmed by qPCR and Western blotting. G100S) is overexpressed at mRNA and protein levels, and this mutation can stably passage in transgenic mice. Conclusions This study successfully established a heart-specific human PRKAG2 (G100S) transgenic mouse model. The role of human PRKAG2 (G100S) gene overexpression in cardiomyocyte development and function maintenance needs to be further studied and discussed .