Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus
Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus
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DOI：
10.1089/scd.2011.0163
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发表时间：
2012-01-01
影响因子：
4
通讯作者：
Liu, Ying
中科院分区：
文献类型：
--
作者：
MacArthur, Chad C.;Xue, Haipeng;Liu, Ying
人类胚胎干细胞 (hESC) 系的谱系报告基因可用于分化研究和药物筛选。此前，我们以位点特异性方式在 hESC 系 WA09 和异常 hESC 系 BG01V 的染色体 13q32.3 基因座上创建了由延伸因子 1 α (EF1 α) 启动子驱动的报告系。这些品系中记者的表达在长期培养中保持在未分化的状态。然而，当这些细胞分化成特定谱系时，观察到报告基因表达减少，表明转基因沉默。为了在 hESC 中开发高效可靠的基因工程策略，我们使用染色质绝缘体元件侧接单拷贝转基因，并通过 PhiC31/R4 整合酶介导的重组技术将组合表达构建体精确整合到 13 号染色体位点。将两个拷贝的 cHS4 双绝缘体序列放置在启动子报告构建体的 5' 和 3' 附近。绿色荧光蛋白 (GFP) 基因由 EF1 α 或 CMV 早期增强子/鸡 β 肌动蛋白 (CAG) 启动子驱动。在工程化 hESC 系中，对于绝缘的 CAG-GFP 和 EF1 α-GFP，在长时间培养和针对不同类型的神经元、胰腺内胚层和中胚层后代的定向分化测定中，染色体 13 基因座的组成型表达得以维持。特别是，这里描述的是第一个正常的 hESC 荧光报告基因系，它在未分化状态和整个多巴胺能谱系分化过程中都强烈表达 GFP。利用绝缘子序列和在 13 号染色体组成型基因座处整合的双重策略确保了适当的转基因表达。这是使用 hESC 进行谱系发育研究、功能获得和丧失实验以及人类疾病建模的宝贵工具。
Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1 alpha) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1 alpha or CMV early enhancer/chicken beta actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1 alpha-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.