摘要
目的:建立一种可诱导稳定表达方法,并构建DLX4细胞系。分析DLX4细胞系的表达调控。方法:将DLX4全长基因克隆入pcDNA5/FRT/TO表达载体,将其转染入宿主细胞Flp-InTMT-RExTM-293细胞系,潮霉素B筛选或者阳性克隆。采用不同剂量的DOX或者不同作用时间对pcDNA5/DLX4细胞系进行诱导表达,用Western blotting检测DLX4表达情况。结果:构建了可被Doxycline(DOX)诱导的稳定表达pcDNA5/DLX4的细胞系。0.001μg/ml的DOX即可诱导pcDNA5/DLX4细胞系的表达,0.1μg/ml DOX即可诱导该细胞系表达达到高峰;DOX对该细胞系作用1h后即可诱导该细胞系表达,对其作用16h后该细胞系表达明显增强。结论:构建了可被DOX诱导稳定表达的DLX4细胞系,不同剂量的DOX和不同作用时间对DLX4细胞系表达有不同的影响。该细胞系的建立为深入探索DLX4的功能提供了一种新的有效方法和实验材料。
Objective： To establish an inducible and stable expression method to make DLX4 cell line,and to analyze the expressive regulation of DLX4 cell line by Doxycline.Methods： The DLX4 ORF was cloned into pcDNA5/FRT/TO expression vector,then transfected into the host cell line Flp-InTMT-RExTM-293,and the positive clones were selected using hygromycin B as selection drug.The expression of pcDNA5/DLX4 cell line was induced with different dosage of DOX or different action time with certain DOX dosage.Western blotting was used to detect the expression of DLX4.Results：We established DLX4 cell line that could stably express DLX4 under the induction of DOX.0.001μg/ml of DOX began to induce the expression of DLX4 protein in the pcDNA5/DLX4 cell line,and the expression level reached the highest with 0.1μg/ml of DOX;the pcDNA5/DLX4 cell line started to express DLX4 protein after DOX treatment for 1 hour,and the expression became stronger apparently with the treatment for 16 hours.Conclusion： We have successfully established the DLX4 cell line that can stably express DLX4 under the induction of DOX,and different dosage of DOX and different action time have different effect on our DLX4 cell line.The establishment of DLX4 cell line provides us an effective method and experimental material for further exploring the DLX4 function.
出处
《泸州医学院学报》
2011年第5期480-482,共3页
Journal of Luzhou Medical College
基金
国家自然基金项目(81172049)
四川省教育厅重点项目(10ZA038)
泸州医学院启动基金资助项目
