Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.
Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.
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DOI：
10.1186/1741-7007-10-93
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发表时间：
2012-11-30
期刊：
影响因子：
5.4
通讯作者：
Mumm JS
中科院分区：
文献类型：
--
作者：
Xie X;Mathias JR;Smith MA;Walker SL;Teng Y;Distel M;Köster RW;Sirotkin HI;Saxena MT;Mumm JS
我们已经研究了一个简单的策略，提高转基因表达的特异性，利用遗传沉默元件。该方法用于将转基因表达限制于感兴趣的组织（在本文提供的实施例中为神经系统），从而促进离散细胞亚型的特异性/排他性靶向。最近的创新使我们更接近了解大脑是如何组织的，神经回路如何运作，以及神经元如何再生。荧光蛋白能够绘制"连接体"，光遗传学工具允许可兴奋细胞短路或过度激活，并且神经元亚型的靶向消融促进电路功能和神经元再生的研究。最佳地，此类工具集需要仅在感兴趣的细胞类型内表达，因为非现场表达使得难以建立因果关系。为了解决这个问题，我们已经开发了一种基因“沉默”系统，通过抑制非神经组织中的表达来促进神经元特异性。该方法解决了困扰大规模增强子捕获工作的非特异性背景问题，并且可以提供利用启动子/增强子的手段，否则启动子/增强子表达太广泛而对体内操作没有价值。我们发现，一个保守的神经元限制性沉默元件（NRSE）可以发挥作用，以限制转基因表达的神经系统。神经元限制性沉默因子/阻遏物元件1沉默转录因子（NRSF/REST）转录阻遏物结合NRSE/阻遏物元件1（RE 1）位点并沉默非神经元细胞中的基因表达。将NRSE位点插入转基因强烈偏向于神经组织表达。NRSE序列在增强子陷阱的背景下并且当与限定的启动子和增强子相关联时，在限制基于二分Gal 4的"驱动"转基因的表达中是有效的。然而，NRSE序列并没有起到限制表达的上游激活序列（UAS）为基础的报告/效应转基因时，仅与UAS元件。Morpholino knockdown分析表明，NRSF/REST表达是基于NRSE的转基因沉默所必需的。我们的研究结果表明，添加NRSE序列的转基因可以为神经系统的功能研究提供有用的新工具。然而，一般的方法可以更广泛地适用;组织特异性沉默元件在神经系统以外的组织中是可操作的，这表明这种方法可以类似地应用于其他范例。因此，在内源性调节元件和组织特异性沉默子之间产生合成关联可以促进靶向缺乏确定的启动子/增强子的细胞亚型。
We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations. We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing. Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.