Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects
Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects
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DOI：
10.1089/hum.2007.021
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发表时间：
2007-04-01
影响因子：
4.2
通讯作者：
Emery, David W.
中科院分区：
文献类型：
--
作者：
Aker, Mari;Tubb, Julie;Emery, David W.
原型染色质绝缘子cHS 4已被证明有效地减少抑制性染色体位置对逆转录病毒载体表达的影响。我们在这里报告的研究，旨在确定最小的鸡过敏位点-4（cHS 4）的序列需要这种活动。使用γ逆转录病毒报告载体和在细胞系和原代小鼠造血祖细胞集落中的表达分析，我们发现，250 bp的核心片段报道含有大部分cHS 4绝缘活性，当单独使用时不能防止沉默，尽管当该片段与790 bp而不是596 bp的间隔区组合时观察到一些屏障活性。类似的研究表明，含有cHS 4增强子阻断活性的90 bp片段的四个拷贝实际上抑制了载体绿色荧光蛋白（GFP）的表达。相反，含有250-bp核心加上3'侧翼序列的400-bp片段保护载体表达至与全长1.2-kb片段相同的程度。在鼠红系白血病（MEL）细胞中表达人β-珠蛋白的慢病毒载体中证实了400-bp片段的活性。总之，这些研究表明，250 bp的cHS 4核心的绝缘活性可以受到距离的影响，并确定了一个扩展的核心元件，赋予两个不同类别的逆转录病毒载体的设置完全的屏障活性。
The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.