心脏过表达人源PRKAG2(G100S)转基因小鼠模型的建立

Establishment of transgenic mouse model with cardiac overexpression of PRKAG2-G100S

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 novel transgenic mouse model of human PRKAG2 cardiac syndrome that overexpresses a PRKAG2-GIOOS mutation, so as to lay a foundation for further studying the role of human PRKAG2 gene in the development, morphology, and function of mouse heart. Methods Human PRKAG2 with G100S mutation was sub-cloned into a multiple cloning site located in the downstream of a-myosin heavy chain（a-MHC） promoter of the plasmid. After the construction of the transgenic expressing vector, C57BL/6J mice were selected as the genetic background, and the transgenic mouse model of PRKAG2-GIOOS mutation was built by microinjection. Genotype was further confirmed using specific primer PCR. Real time PCR and Western blotting analysis were used to examin the expression of human PAKAG2 （G100S） mRNA and protein, respectively. Results Two strains of transgenic mice were successfully developed using backcross breeding, which specifically overexpressed the human PRKAG2-G100S mutation in the cardiac tissues of F2 generations by the methods qPCR and Western blotting at both mRNA and protein levels. Moreover, the PRKAG2-GIOOS mutation was successfully passed steadily. Conclusion We have successfully established a human PRKAG2-GIOOS transgenic mouse model, which can help to further explore the role of PRKAG2-GIOOS mutation in the development and function of mouse cardiac tissue in the PRKAG2- G100S cardiac syndrome.