组蛋白甲基化抑制剂UNC0638对水牛转基因细胞外源基因表达的影响

Effect of the Histone Methylation Inhibitor UNC0638 on Exogenous Genes Expression of Transgenic Buffalo Fibroblast

本研究旨在探讨不同浓度的组蛋白甲基化抑制剂UNC0638对转基因水牛成纤维细胞外源基因表达水平的影响,为外源基因表达机制研究及提高转基因水牛生产效率提供理论依据。试验首先使用不同浓度的UNC0638(0(对照组)、0.5、1.5、2.5、5.0μmol·L^(-1))处理水牛胎儿成纤维细胞(BFFs)8d,绘制细胞生长曲线,并在UNC0638(0(对照组)、0.5、1.5、2.5μmol·L^(-1))处理BFFs 48h后,检测细胞核型,同时利用细胞免疫荧光技术分析组蛋白H3K9me2甲基化水平,采用实时荧光定量PCR(qRT-PCR)检测UNC0638对水牛转基因细胞外源基因表达效率的影响。结果表明:1)BFFs经不同浓度UNC0638处理后,生长曲线与对照组相似,均呈"S"形分布,0.5、1.5、2.5μmol·L^(-1 )UNC0638处理组与对照组相比细胞增殖效率没有发生显著变化,5.0μmol·L^(-1)UNC0638抑制BFFs增殖;2)各处理组细胞核型正常率与对照组间差异不显著;3)此外,UNC0638处理组的细胞组蛋白H3K9me2甲基化水平显著低于对照组(P<0.05);4)UNC0638(0、0.5、1.5、2.5μmol·L^(-1))处理水牛转基因胎儿成纤维细胞24h,2.5μmol·L^(-1)组细胞eGFP的相对表达量显著高于对照组(P<0.05);处理48h,0.5、1.5、2.5μmol·L^(-1)组eGFP的表达量与对照组相比显著提高(P<0.05)。综上表明,UNC0638可有效降低水牛成纤维细胞组蛋白H3K9me2甲基化水平并提高水牛转基因细胞外源基因的表达效率。

The purpose of this study was to evaluate the effect of different concentrations of the histone methylation inhibitor UNC0638 on the expression level of transgenic buffalo fibroblast exogenous gene to provide a theoretical basis for studying the mechanism of exogenous gene expression and improving the production efficiency of transgenic buffaloes.After the buffalo fetal fibroblasts(BFFs)were treated with different concentrations of UNC0638(0(control group),0.5,1.5,2.5,5.0 mol·L^-1)for 8 d,the cell growth curve was plotted and the cell karyotype was detected after treated with different concentrations of UNC0638 for 48 h.The methylation level of the histone H3K9me2 of BFFs was detected by immunofluorescent technique,and the effect of UNC0638 on the expression efficiency of buffalo transgenic cells exogenous genes was analyzed by quantitative real-time PCR(qRT-PCR).The results showed that:1)The growth curves of BFFs treated with different concentrations of UNC0638 were similar to that of the control group and all showed"S"type,and 0.5,1.5,2.5μmol·L^-1 UNC0638 had no significant effect on the cell proliferation,while 5.0μmol·L^-1 UNC0638 inhibited the BFFs proliferation.2)No significant difference was observed in the normal rate of the cell karyotype among the different groups.3)In addition,the methylation level of histone H3K9me2 in BFFs treated with UNC0638 was significantly decreased compared with the control group(P<0.05).4)The qRT-PCR results showed that the relative expression of eGFP in the buffalo transgenic fetal fibroblasts treated with 2.5μmol·L^-1 UNC0638 was significantly higher than that in the control group in 24 h(P<0.05),while the 0.5,1.5 and 2.5μmol·L^-1 UNC0638 groups were significantly higher than that in the control group in 48 h(P<0.05).In conclusion,the research showed that UNC0638 could effectively reduce the methylation level of the buffalo fibroblast histone H3K9me2 and increase the efficiency of exogenous gene expression in buffalo transgenic cells.