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目的:构建以绿色荧光蛋白(GFP)为报告基因的重组表达质粒pEGFP-C1-PPARγ,观察小鼠PPARγ基因在MDA-MB-231细胞中的表达及定位。方法:采用克隆和亚克隆技术构建小鼠PPARγ基因真核表达载体,脂质体Lip2000介导转染MDA-MB-231细胞,real-time PCR和western-blot验证其mRNA和蛋白的表达,荧光显微镜观察该基因亚细胞定位。结果:酶切和测序结果证实重组质粒含有PPARγ编码区序列且插入方向正确,转染后观察该基因亚细胞定位于胞核,胞质有弥散分布。结论:成功构建了小鼠PPARγ基因真核表达载体,该基因在MDA-MB-231细胞中成功表达,PPARγ基因主要集中表达于胞核。
OBJECTIVE: To construct a recombinant plasmid pEGFP-C1-PPARγ with green fluorescent protein (GFP) as reporter gene and to observe the expression and localization of mouse PPARγ gene in MDA-MB-231 cells. Methods: The mouse PPARγ gene eukaryotic expression vector was constructed by cloning and subcloning techniques. Lipofectamine 2000 was transfected into MDA-MB-231 cells by lipofectamine 2000. The mRNA and protein expression of PPARγ gene was detected by real-time PCR and western-blot. Microscopic observation of the subcellular localization of the gene. Results: The result of restriction enzyme digestion and sequencing confirmed that the recombinant plasmid contained the PPARγ coding region and inserted correctly. After transfection, the subcellular localization of the gene was located in the nucleus and the cytoplasm was diffusely distributed. Conclusion: The mouse PPARγ gene eukaryotic expression vector was successfully constructed. The gene was successfully expressed in MDA-MB-231 cells. The PPARγ gene was mainly expressed in the nucleus.