应用绿色荧光研究人抑瘤素基因在CHO细胞中的表达

目的：建立一种简易、快速检测细胞内外源性基因稳定表达的方法。方法：以携带绿色荧光蛋白的真核表达质粒pEGFP-N1为骨架，将完整的 hOSM基因ORF克隆到pEGFP-N1上游的多克隆位点，构建表达载体pOSM-EGFP-N1，脂质体介导转染中国仓鼠卵巢细胞（CHO），G418筛选，挑选阳性克隆。荧光显微镜、流式细胞术分析转染了pOSM-EGFP-N1质粒的CHO细胞纯度。RT-PCR的方法分析OSM基因水平表达。结果：成功建立稳定表达OSM的CHO细胞株（CHO-OSM-EGFP），纯度达到99.76%，且OSM基因在CHO-OSM-EGFP细胞中稳定表达。结论：构建了携带绿色荧光、表达OSM的pOSM-EGFP-N1质粒转染的CHO细胞，只通过荧光显微镜观察和FCM检测细胞绿色荧光，即可明确OSM转染成功。为表达外源性基因阳性细胞的筛选提供了简易的方法。

Employing green fluorescence to investigate expression of human Oncostatin M in CHO cell line

Objective:To establish a simple and rapid method for measuring the exogenous gene expression in eukaryotic cells.Methods: The plasmid that expresses green fluorescence,pOSM-EGFP-N1 was constructed by the insertion of the complete ORF of human oncostatin M (OSM) into pEGFP-N1 vector,and transfect Chinese hamster ovary(CHO) cells to get CHO-OSM-EGFP cells.After screening with G418 culture,the colony of expression green fluorescence was picked up under the fluorescence microscope.The CHO-OSM-EGFP cell line was obtained by continuously cultivation.Purity of cell line was checked by flow(cytometry)(FCM).The OSM mRNA expression was examined by RT-PCR.Results:The CHO-OSM-EGFP cell line is stable expresses green fluorescence and the cell colonies with uniformly fluorescent intensity in culture and easily observed by fluorescence microscope.The purity of the CHO-OSM-EGFP cell line is 99.76% that analysis by FCM.The target gene OSM expression was detected in CHO-OSM-EGFP cell line by RT-PCR.Conclusion:These results showed that inserting the OSM gene into an expression green fluorescence vector,pEGFP-N1.The plasmid pOSM-EGFP-N1 transfect CHO cells,we can get stable expresses green fluorescence CHO-OSM-EGFP cell line.Through the green fluorescence expression,we will identify the exogenous gene expression in the eukaryotic cells.oncostatin M;fluorescence;gene expression;CHO cell