【摘要】 目的:构建p-EGFP-C3-Wnt-1真核表达载体并转染体外培养大鼠成肌细胞,观察Wnt-1在其中的表达。方法:利用RT-PCR方法从小鼠胚脑中扩增获得Wnt-1基因编码区全长序列,克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体p-EGFP-C3上,形成重组载体p-EGFP-C3-Wnt-1。大鼠成肌细胞原代培养及desmin免疫荧光染色鉴定。利用LipofectamineTM2000脂质体将重组质粒p-EGFP-C3-Wnt-1转染成肌细胞。结果:重组质粒p-EGFP-C3-Wnt-1构建成功;将其转染成肌细胞72h后,观察到EGFP报告基因和目的基因Wnt-1表达。结论:成功构建了小鼠胚脑Wnt-1基因的真核表达载体p-EGFP-C3-Wnt-1,并可转染成肌细胞。更多还原

【Abstract】 Objective To construct mouse Wnt-1 gene eukaryotic expression vector and to detect its expression in rat skeletal myoblasts in vitro.Methods Mouse Wnt-1 gene was amplified from the brain tissue of fetal mouses by RT-PCR,and was inserted into an eukaryotic expression vector p-EGFP-C3 carrying the enhanced green fluorescent protein,to generate a new plasmid p-EGFP-C3-Wnt-1.Skeletal myoblasts were cultured and identified by desmin immunofluorescence staining.The identified recombinate p-EGFP-C3-Wnt-1 eukaryotic expression vector was transfected into rat skeletal myoblasts with Lipofectamine TM2000 liposome.After transfection for 72h,p-EGFP-C3-Wnt-1 expression was examined in skeletal myoblasts.Results The eukaryotic expression vector containing mouse Wnt-1 gene was constructed successfully.EGFP report gene and Wnt-1 gene were expressed in myoblasts transfected with the recombinate plasmid p-EGFP-C3-Wnt-1.Conclusion The recombinant vector p-EGFP-C3-Wnt-1 is constructed and mouse Wnt-1 gene can be normally expressed in skeletal myoblasts.更多还原