Tet调控的脑相对特异性新基因LRRC4表达细胞系的构建  

Establishment of Brain Relatively Specific Gene LRRC4 With Doxycycline Induced Tet Regulating System in U251 Glioblatoma Cell Line

作　　者：张秋红[1] 王莉莉[1] 彭聪[1] 曹利[1] 王洁如[1] 李小玲[1] 李桂源[1] 

机构地区：[1]中南大学肿瘤研究所,长沙410078

出　　处：《生物化学与生物物理进展》2005年第4期325-330,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目( 30100191.30270429);湖南省自然科学基金资助项目(03JJY3062).~~

摘　　要：LRRC4是一个在脑相对特异性表达的富亮氨酸重复超家族新成员,在神经胶质瘤表达明显下调或缺失且具有抑制脑胶质瘤细胞生长的潜能. 利用Tet-on基因表达系统,经过两轮转染,先后将调控质粒pTet-on和表达质粒pTRE-2hyg-LRRC4转染U251细胞系,分别用G418和潮霉素Hygromycin进行两次筛选. 在第一轮挑取的80个克隆中,利用pTRE-2hyg-luciferase报告基因进行最佳的低背景高表达的pTet-on细胞克隆筛选,在通过量效关系和动力学检测筛选的最佳克隆基础上,再进行pTRE-2hyg-LRRC4的转染,并通过RT-PCR和RNA印迹检测,成功获得了两个具有良好诱导性Tet调控的LRRC4双稳定表达细胞系,为进一步阐明LRRC4在脑胶质瘤发生发展中的作用,提供有利的研究基础和理想的实验平台.LRRC4 is a novel brain relatively specific gene and a member of LRR superfamily, which displayed significant down-regulation in primary brain tumor biopsies and has the potential to suppress brain tumor growth. The establishnent of LRRC4 with doxycycline (Dox) induced Tet regulating system in U251 glioblastoma cell line was reported. Firstly, Tet-on regulating plasmid was transfected into U251 cells and screened by 6418 to construct the single-stable U251 Tet-on cell line. Low background and high expression clone was acquired by testing luciferase activity. Successively, pTRE-2hyg/LRRC4 plasmid was transfected into the clone and screened by hygromycin. Two positive clones were received by RT-PCR and Northern blot analysis. The positive clones showed well dose-response and time-response in expression of LRRC4 with Dox inducement. Results show that establishment of LRRC4 with doxycycline induced Tet regulating system in U251 glioblatoma cell line is successfully, which provides an ideal experimental platform for understanding the mechanism of LRRC4 in glioma tumorigenesis and development.

关 键 词：LRRC4 Tet-on基因表达系统 DOXYCYCLINE 

分 类 号：R73-3[医药卫生—肿瘤]