利用高压水枪法将双报告基因导入小鼠肝脏

Abstract

目的：通过高压水枪法将双报告基因[即绿色荧光蛋白(GFP)和萤火虫荧光素酶(Luc)]导入小鼠肝脏，以熟练掌握高压水枪法，其将为后续相关研究奠定方法学基础。方法：将质粒pcEF.luc-IRES-GFP瞬时转染入人肝癌细胞SMMC-7721以进行载体体外功能验证，并利用高压水枪法将质粒pcEF.luc-IRES-GFP经尾静脉注射入小鼠肝脏内，经小动物活体成像系统活体检测GFP和Luc信号，同时免疫组化检测GFP在肝组织中的表达情况。结果：瞬转结果显示，7721细胞上可检测到GFP和Luc表达；利用高压水枪法将pcEF.luc-IRES-GFP导入小鼠肝脏后，利用小动物活体成像仪成功在肝内检测到GFP和Luc信号；免疫组化结果显示部分肝细胞表达GFP。结论：成功掌握了将目的基因导入小鼠肝脏的高压水枪法，这为相关后续研究打下良好基础。 Objective: Set up the system for hydrodynamic transfection of double reporter genes [green fluo-rescent protein (GFP) and firefly luciferase (Luc)] into mouse liver. Methods: To in vitro verify the functionality of pcEF.luc-IRES-GFP, human hepatocellular carcinoma 7721 cells were transiently transfected with pcEF.luc-IRES-GFP, followed by GFP and Luc assay. Hydrodynamic transfection was employed to deliver pcEF.luc-IRES-GFP into mice via tail-vein injection, followed by in vivo and ex vivo imaging to detect Luc and GFP. Moreover, immunohistochemistry (IHC) were performed to detect GFP expression in mouse liver cells. Results: The expression of GFP and Luc was detected in 7721 cell transiently transfected by pcEF.luc-IRES-GFP. Luc signals were detected in liver by in vivo bioluminescence imaging, and both Luc and GFP were detected in isolated liver by in vivo bioluminescence and fluorescence imaging. IHC revealed the GFP-positive liver cells of mice hydrodynamically transfected via tail-vein injection of pcEF.luc-IRES-GFP. Conclusions: We successfully deliver double reporter genes (GFP and luciferase) into mouse liver by hydrodynamic transfection, which lays a solid foundation for further research.