启动子上的增强子和CpG岛在转基因沉默和位置效应中的作用  

Roles of enhancers and CpG islands on promoters in transgene silencing and position effect

作　　者：肖捷 纪华 王宵 王静[3] 刘凌云[3] 李美荃 王斌[3] XIAO Jie;JI Hua;WANG Xiao;WANG Jing;LIU Lingyun;LI Meiquan;WANG Bin(College of Pharmacy,Dali University,Dali 671000,Yunnan Province,China;Department of Oncology,the First People′s Hospital of Yunnan Province,Kunming 650034,Yunnan Province,China;Engineering Research Center of New Livestock and Poultry Vaccine and Key Industry Technology,Yunnan Education Department,Kunming University,Kunming 650214,Yunnan Province,China)

机构地区：[1]大理大学药学院,云南大理671000 [2]云南省第一人民医院肿瘤科,云南昆明650034 [3]昆明学院云南教育厅新型畜禽疫苗及产业关键技术工程研究中心,云南昆明650214

出　　处：《新乡医学院学报》2023年第9期801-809,共9页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:32760256,32060147,21967019);联勤保障部队第920医院基金(编号:2019YGB22);云南省地方高校联合项目(编号:202001BA070001-217)。

摘　　要：目的探讨哺乳动物细胞表达载体启动子上增强子和CpG岛调控元件在转基因沉默和位置效应中的作用。方法应用带增强子的八聚体结合转录因子4(OCT4)基因启动子、带CpG岛的性别决定区Y框蛋白2(SOX2)基因启动子、带增强子和CpG岛的巨细胞病毒(CMV)基因启动子以及不含近端增强子和CpG岛的NANOG基因启动子构建哺乳动物细胞表达载体,并分别转染中国仓鼠卵巢细胞株CHO-K1细胞和人胚胎肾细胞(HEK293)。应用Image J软件中Mean算法计算OCT4、SOX2、CMV和NANOG基因启动子在CHO-K1细胞和HEK293细胞介导的增强绿色荧光蛋白(EGFP)表达的荧光强度,应用Image J软件中Mininum、Maxentropy、Mean 3种算法分别计算OCT4、SOX2、CMV和NANOG基因启动子4种载体稳定转染CHO-K1细胞后形成的多个单细胞克隆混合生长样品在同一个样本中最大荧光强度的细胞、大于平均荧光强度的所有细胞和最小荧光强度的所有细胞的EGFP平均荧光强度;应用有限稀释法从G418筛选后获得的稳定转染的多个单克隆混合的CHO-K1细胞中,分别筛选含EGFP的pE-C1、pE-Oct4、pE-Sox2、pE-Nanog质粒稳定转染CHO-K1细胞的单克隆细胞,每个质粒载体随机筛选3个单克隆细胞,应用Image J软件分析EGFP平均荧光强度;应用酶联免疫吸附法检测转染第20、30天CHO-K1细胞中EGFP蛋白表达水平,应用实时荧光定量聚合酶链式反应法检测转染第20、30天CHO-K1细胞中EGFP mRNA表达水平。结果SOX2基因启动子在CHO-K1细胞和HEK293细胞中介导表达的EGFP平均荧光强度比较差异无统计学意义(t=0.770,P>0.05)。OCT4、CMV、NANOG基因启动子在CHO-K1细胞中介导表达的EGFP平均荧光强度显著高于HEK293细胞(t=7.000、11.100、4.900,P<0.05)。SOX2基因启动子于转染第20、30天在CHO-K1细胞中介导表达的EGFP蛋白水平比较差异无统计学意义(t=0.330,P>0.05)。CMV基因启动子于转染第20天在CHO-K1细胞中介导表达的EGFP蛋�Objective To explore the role of enhancers and CpG island regulatory elements on the promoter in transgenic silencing and positional effects in mammalian cell expression vectors.Methods Mammalian cell expression vectors were constructed by using the octamer binding transcription factor 4(OCT4)gene promoter with enhancer,the sex determining region Y box protein 2(SOX2)gene promoter with CpG island,the cytomegalovirus(CMV)gene promoter with enhancer and CpG island,and the NANOG gene promoter without proximal enhancer and CpG island,and they were transformed into CHO-K1 cells and human embryonic kidney 293(HEK293)cells,respectively.The fluorescence intensity of enhanced green fluorescent protein(EGFP)mediated by OCT4,SOX2,CMV and NANOG gene promoter in CHO-K1 and HEK293 cells was calculated by using the Mean algorithm in Image J software;the mean fluorescence intensity of EGFP in the all cells with the strongest fluorescence intensity in the same sample,all cells with greater than the average fluorescence intensity and all cells with the smallest fluorescence intensity of multiple single cell clones of CHO-K1 cells formed by stable transfection of OCT4,SOX2,CMV and NANOG gene promoter were calculated by using Mininum,Maxentropy and Mean algorithms in Image J software,respectively.pE-C1,pE-Oct4,pE-Sox2 and pE-Nanog plasmid stably transfected monoclonal cells was screened by using limited dilution method from the stable transfected multiple monoclonal mixture of CHO-K1 cells obtained after G418 screening,and three monoclonal cells were randomly screened in each plasmid vector,and the mean fluorescence intensity of EGFP was analyzed by Image J software;the expression level of EGFP protein in CHO-K1 cells on the 20 th and 30 th day of transfection was detected by using enzyme-linked immunosorbent assay,and the expression level of EGFP mRNA in CHO-K1 cells on the 20 th and 30 th day of transfection was detected by using real-time fluorescence quantitative polymerase chain reaction.Results There was no statistically signi

关 键 词：增强子 CPG岛 转基因沉默 位置效应 哺乳动物细胞表达载体 

分 类 号：R31[医药卫生—基础医学]