Tet 调控的脑相对特异性新基因 LRRC4表达细胞系的构建

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.

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

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 G418 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.