[1]肖捷,纪华,王宵,等.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[J].新乡医学院学报,2023,40(9):801-809.[doi:10.7683/xxyxyxb.2023.09.001]
　XIAO Jie,JI Hua,WANG Xiao,et al.Roles of enhancers and CpG islands on promoters in transgene silencing and position effect[J].Journal of Xinxiang Medical University,2023,40(9):801-809.[doi:10.7683/xxyxyxb.2023.09.001]
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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
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
40卷
期数:
2023年9
页码:
801-809
栏目:
基础研究
出版日期:
2023-09-05

文章信息/Info

Title:
Roles of enhancers and CpG islands on promoters in transgene silencing and position effect
作者:
肖捷1; 纪华2; 王宵3; 王静3; 刘凌云3; 李美荃3; 王斌3
（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）
Author(s):
XIAO Jie1; JI Hua2; WANG Xiao3; WANG Jing3; LIU Lingyun3; LI Meiquan3; WANG Bin3
(1.College of Pharmacy,Dali University,Dali 671000,Yunnan Province,China;2.Department of Oncology,the First People′s Hospital of Yunnan Province,Kunming 650034,Yunnan Province,China;3.Engineering Research Center of New Livestock and Poultry Vaccine and Key Industry Technology,Yunnan Education Department,Kunming University,Kunming 650214,Yunnan Province,China)
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
Keywords:
enhancer; CpG island; transgene silencing; position effect; mammalian cell expression vector
分类号:
R31
DOI:
10.7683/xxyxyxb.2023.09.001
文献标志码:
A
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蛋白水平显著低于转染第30天（t=3.770，P<0.05）；OCT4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=2.500，P>0.05）；NANOG基因启动子于转染第20、30天在CHO-K1细胞中介导表达的EGFP蛋白表达水平比较差异无统计学意义（t=0.014，P>0.05）。转染第20天与转染第30天SOX2、CMV、NANOG基因启动子在CHO-K1细胞中介导表达的EGFP平均荧光强度值比较差异均无统计学意义（t=0.130、0.830、0.210，P>0.05）；转染第30天，OCT4基因启动子在CHO-K1细胞中介导表达的EGFP平均荧光强度值显著低于转染第20天（t=5.750，P<0.05）。SOX2、CMV、OCT4、NANOG基因启动子在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（F=4.070，P>0.05）。CMV、OCT4、NANOG基因启动子在CHO-K1细胞中介导的EGFP mRNA相对表达量显著高于SOX2基因启动子（t=5.440、5.000、5.740，P<0.05）；CMV、OCT4基因启动子在CHO-K1细胞中介导的EGFP mRNA相对表达量显著高于NANOG基因启动子（t=3.220、4.270，P<0.05）；CMV基因启动子在CHO-K1细胞中介导的EGFP mRNA相对表达量高于OCT4基因启动子，但差异无统计学意义（t=1.270，P>0.05）。SOX2基因启动子介导表达的EGFP在转录水平和转录后水平差异最大（t=16.900，P<0.05）；NANOG基因启动子介导表达的EGFP在转录水平和转录后水平差异次之（t=14.930，P<0.05）；OCT4和CMV基因启动子介导表达的EGFP在转录水平和转录后水平差异最小（t=2.060、0.430，P>0.05）。应用Mininum、Maxentropy、Mean 3种算法计算OCT4基因启动子在多克隆下介导表达的EGFP的荧光强度比较差异无统计学意义（F=3.720，P>0.05），SOX2、CMV和NANOG基因启动子在多克隆下介导EGFP表达的荧光强度比较差异均有统计学意义（F=516.400、428.500、28.120，P<0.05）。不同单克隆细胞中的差异分析显示，4种载体在不同单克隆细胞中介导表达的 EGFP 荧光强度值的标准误差相比，从高到低依次为NANOG基因启动子在不同单克隆细胞中介导表达的EGFP荧光强度值标准误差、SOX2基因启动子在不同单克隆细胞中介导表达的EGFP荧光强度值标准误差、OCT4基因启动子在不同单克隆细胞中介导表达的EGFP荧光强度值标准误差、CMV基因启动子在不同单克隆细胞中介导表达的EGFP荧光强度值标准误差，且OCT4基因启动子与SOX2、CMV基因启动子在单克隆细胞中介导表达的EGFP平均荧光强度比较差异有统计学意义（t=3.070、4.360，P<0.05）。
结论　带CpG岛的启动子能在转录后克服转基因沉默效应，带增强子的启动子具有克服位置效应的作用；二者的恰当组合可用于设计在哺乳动物细胞中高效稳定表达的启动子。
Abstract:
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 20th and 30th day of transfection was detected by using enzyme-linked immunosorbent assay,and the expression level of EGFP mRNA in CHO-K1 cells on the 20th and 30th day of transfection was detected by using real-time fluorescence quantitative polymerase chain reaction.
Results　There was no statistically significant difference in the mean fluorescence intensity of EGFP mediated by the SOX2 gene promoter between CHO-K1 cells and HEK293 cells (t=0.770,P>0.05);the mean fluorescence intensity of EGFP mediated by the OCT4,CMV and NANOG gene promoters in CHO-K1 cells was significantly higher than that in HEK293 cells (t=7.000,11.100,4.900;P<0.05).There was no statistically significant difference in EGFP protein level mediated by SOX2 gene promoter in CHO-K1 cells on the 20th and 30th day of transfection (t=0.330,P>0.05);the level of EGFP protein mediated by CMV gene promoter in CHO-K1 cells on the 20th day of transfection was significantly lower than that on the 30th day of transfection (t=3.770,P<0.05);there was no statistically significant difference in EGFP protein level mediated by the OCT4 gene promoter in CHO-K1 cells on the 20th and 30th day of transfection (t=2.500,P>0.05);there was no statistically significant difference in EGFP protein level mediated by the NANOG gene promoter in CHO-K1 cells on the 20th and 30th day of transfection (t=0.014,P>0.05).There was no statistically significant difference in the mean fluorescence intensity of EGFP mediated by SOX2,CMV,NANOG gene promoter in CHO-K1 cells between the 20th and 30th day of transfection (t=0.130,0.830,0.210;P>0.05);the mean fluorescence intensity of EGFP mediated by the OCT4 gene promoter in CHO-K1 cells on the 30th day of transfection was significantly lower than that on the 20th day of transfection (t=5.750,P<0.05).There was no statistically significant difference in EGFP protein level mediated by SOX2,CMV,OCT4,NANOG gene promoters in CHO-K1 cells (F=4.070,P>0.05).The relative expression level of EGFP mRNA mediated by CMV,OCT4,NANOG gene promoters was significantly higher than that of SOX2 gene promoter in CHO-K1 cells (t=5.440,5.000,5.740;P<0.05);the relative expression level of EGFP mRNA mediated by CMV and OCT4 gene promoters was significantly higher than that of NANOG gene promoter in CHO-K1 cells (t=3.220,4.270;P<0.05);the relative expression level of EGFP mRNA mediated by CMV gene promoter was significantly higher than that of OCT4 gene promoter in CHO-K1 cells,but the difference was not significant (t=1.270,P>0.05).The expression of EGFP mediated by SOX2 gene promoter showed the greatest difference at the transcriptional and posttranscriptional levels (t=16.900,P<0.05),followed by the EGFP expression mediated by NANOG gene promoter at the transcriptional and posttranscriptional levels (t=14.930,P<0.05),and the expression of EGFP mediated by OCT4 and CMV gene promoters showed the smallest difference at the transcriptional and posttranscriptional levels (t=2.060,0.430;P>0.05).There was no statistically significant difference in the fluorescence intensity of EGFP mediated by OCT4 gene promoter under polyclonal conditions calculated by using Minimum,Maxentropy,and Mean(F=3.720,P>0.05).However,there were statistically significant differences in the fluorescence intensity of EGFP expression mediated by SOX2,CMV,NANOG gene promoters under polyclonal conditions (F=516.400,428.500,28.120;P<0.05).The difference analysis in different monoclone cells showed that the standard error of EGFP fluorescence intensity of the four vectors in different monoclone cells from high to low was the fluorescence intensity standard error of NANOG gene promoter mediated EGFP expression in different monoclonal cells,the fluorescence intensity standard error of SOX2 gene promoter mediated EGFP expression in different monoclonal cells,the standard error of fluorescence intensity value of OCT4 gene promoter mediated EGFP expression in different monoclone cells,and the standard error of fluorescence intensity value of CMV gene promoter mediated EGFP expression in different monoclone cells.And there were statistically significant differences in the mean fluorescence intensity of EGFP of monoclone cells between OCT4 gene promoter and SOX2,CMV gene promoters(t=3.070,4.360;P<0.05).
Conclusion　The promoter containing CpG islands can overcome transgene silencing effect after transcription,the promoters containing enhancers can overcome the position effect.The appropriate combination of CpG islands and enhancers can be used to design promoters for efficient and stable expression in mammalian cells.

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更新日期/Last Update: 2023-09-05