Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats
Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats
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DOI：
10.4238/gmr.15038560
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发表时间：
2016-01-01
影响因子：
0.4
通讯作者：
Liu, D. J.
中科院分区：
文献类型：
--
作者：
Nuo, M. T.;Yuan, J. L.;Liu, D. J.
转基因沉默常见于转基因动植物中，限制了转基因生物的产生和应用，与外源基因拷贝数、启动子甲基化状态、组蛋白修饰异常有关。在这里，我们分析了六只健康转基因绒山羊和转基因核供体细胞中外源基因 DsRed 的表达及其巨细胞病毒（CMV）启动子的甲基化状态。其中四只山羊的 CMV 启动子表现出高甲基化水平（74.4-88.2%），一只山羊表现出中等甲基化水平（58.7%），一只山羊表现出低甲基化水平（21.2%），而转基因核供体细胞的甲基化水平相对较低（14.3%）。 DsRed 表达与启动子甲基化状态呈负相关。转基因绒山羊携带1~3个拷贝的CMV启动子片段和1~6个拷贝的DsRed片段，但拷贝数与DsRed表达无明显相关性。经甲基化抑制剂5-氮杂胞苷处理后，转基因山羊细胞中DsRed表达显着升高，CMV启动子甲基化显着降低；这表明启动子甲基化状态与红色荧光蛋白表达之间呈负相关。用组蛋白脱乙酰酶抑制剂曲古抑菌素A处理后，DsRed表达增加，表明转基因山羊中异常的组蛋白修饰也参与了外源基因沉默。这些发现表明，曲古抑菌素 A 和 5-氮杂胞苷有可能在培养条件下挽救成体来源的转基因细胞中沉默的外源转基因的生物活性。
Transgene silencing, which is common in transgenic plants and animals, limits the generation and application of genetically modified organisms, and is associated with the exogenous gene copy number, the methylation status of its promoters, and histone modification abnormalities. Here, we analyzed the expression of the exogenous gene DsRed and the methylation status of its cytomegalovirus (CMV) promoter in six healthy transgenic cashmere goats and transgenic nuclear donor cells. The CMV promoter exhibited high methylation levels (74.4-88.2%) in four of the goats, a moderate methylation level (58.7%) in one, and a low methylation level (21.2%) in one, while the methylation level of the transgenic nuclear donor cells was comparatively low (14.3%). DsRed expression was negatively correlated with promoter methylation status. Transgenic cashmere goats carried one to three copies of the CMV promoter fragment and one to six copies of the DsRed fragment, but copy number showed no obvious correlation with DsRed expression. After treatment with the methylation inhibitor 5-azacytidine, DsRed expression in transgenic goat cells significantly increased and CMV promoter methylation significantly decreased; this indicated an inverse correlation between promoter methylation status and DsRed expression. After treatment with the histone deacetylase inhibitor trichostatin A, DsRed expression increased, indicating that an abnormal histone modification in transgenic goats is also involved in exogenous gene silencing. These findings indicate the potential of trichostatin A and 5-azacytidine to rescue the biological activity of silenced exogenous transgenes in adult-derived transgenic cells under culture conditions.