Lipofectamine ^(TM)2000介导pIRES2-EGFP-NT3转染新生小鼠离体耳蜗基底膜的实验研究

pIRES2-EGFP-NT3 transfected cultivation of basal membrane of Corti's organ from neonatal mouse in vitro with Lipofectamine 2000

目的构建真核表达质粒载体pIRES2-EGFP-NT3,检测其在阳离子脂质体介导下对新生小鼠离体耳蜗基底膜的转染情况。方法构建含有目的基因NT3的真核表达质粒载体pIRES2-EGFP-NT3后,在阳离子脂质体LipofectamineTM2000介导下将其转染新生小鼠离体耳蜗基底膜培养组织,转染后24 h,通过Confocal显微镜观察小鼠离体耳蜗基底膜的转染情况和转染效率。结果成功构建了真核表达质粒载体pIRES2-EGFP-NT3,通过阳离子脂质体LipofectamineTM2000介导,该质粒在新生小鼠离体耳蜗基底膜转染的范围内转染进17个细胞,说明该质粒在阳离子脂质体介导下能转染新生小鼠离体耳蜗基底膜培养组织。结论含有目的基因NT3的真核表达质粒载体pIRES2-EGFP-NT3的成功构建,及其在阳离子脂质体介导下对新生小鼠离体耳蜗基底膜培养组织的有效转染,为研究NT3基因转染对正常及致聋小鼠耳蜗效应的实验奠定了基础。

Objective To construct pIRES2-EGFP-NT3 and detect its transfection in the in vitro cultivation of the basal membrane of Corti＇s organ from neonatal mouse.Methods After successful construction of pIRES2-EGFP-NT3,the in vitro cultivation of Corti＇s organ from neonatal mouse was transfected by pIRES2-EGFP-NT3 with the mediation of Lipofectamine TM2000.The transfection efficiency was analysed under confocal microscope 24 hours later.Results The pIRES2-EGFP-NT3 was successfully constructed and properly transfected into 17 cells of the basal membrane of Corti＇s organ which was cultivated in vitro with the mediation of cationic liposome.Conclusion The successful construction of pIRES2-EGFP-NT3 and its efficient transfection into cultivation of Corti＇s organ from neonatal mouse by LipofectamineTM2000 lay the groundwork for the following experiments,such as NT3 gene transfection in deaf or nomal cochlea.