稳定整合靶向EGFL7基因的shRNA载体的胶质瘤细胞系的建立  

Development of Stable Glioma Cell Line Expressing shRNA Targeting EGFL7 Gene

作　　者：刘清流[1] 张峰[1] 

机构地区：[1]广州医学院第二附属医院神经外科,广东广州510260

出　　处：《中国医药科学》2011年第9期41-42,共2页China Medicine And Pharmacy

摘　　要：目的构建针对EGFL7基因的特异性RNA干扰载体,建立稳定转染该干扰载体的人胶质瘤细胞系。方法根据EGFL7基因的编码序列设计并合成针对EGFL7基因的特异性shRNA,将其克隆入pSilencer3.1-H1neo载体中,重组载体经脂质体LipofectamineTM2000介导转染胶质瘤细胞株U251;转染细胞经G418筛选,采用Westernblot方法在蛋白水平检测筛选出的G418抗性的克隆细胞。结果酶切鉴定和测序证实,成功构建了靶向EGFL7基因的shRNA真核表达载体pSilencer-shEGFL7,并获得了稳定表达靶向EGFL7基因的shRNA的胶质瘤细胞株。结论 EGFL7基因特异性RNA干扰载体能够显著抑制EGFL7基因在U251细胞中的表达,这为进一步研究EGFL7在胶质瘤细胞系U251中的生物学功能和作用机制奠定了基础。Objective To construct the short hairpin RNA targeting human epidermal growth factor like-7（EGFL7）and develop the stable glioma cell line expressing this shRNA.Methods Based on the EGFL7 gene sequence,shRNA was designed,synthesized and subcloned into the pSilencer 3.1-H1 neo vector.Then the recombinant vector was transfected into U251 cell line and the cell was screened with G418 and assayed by using Western blot.Results Restriction endonuclease digestion test and sequencing vertification showed that the recombinant pSilencer-shEGFL7 vector expressing shRNA targeting EGFL7 was succeccfully constructed and the stable glioma cell line expressing this shRNA was developed.Conclusion Vector expressing shRNA targeting EGFL7 may be used to specially inhibit the expression and furhter investigate the function of EGFL7 gene in the U251 cell line.

关 键 词：人类表皮生长因子域7 SHRNA RNA干扰 U251 胶质瘤 

分 类 号：R730.54[医药卫生—肿瘤]