A medium-scale assay for enhancer validation in amniotes.
A medium-scale assay for enhancer validation in amniotes.
复制标题
DOI：
10.1002/dvdy.24306
复制
发表时间：
2015-10
期刊：
影响因子：
--
通讯作者：
Streit A
中科院分区：
文献类型：
--
作者：
Chen J;Streit A
增强子是在时间和空间上控制基因表达的关键元件，从而在发育、稳态和疾病期间协调基因功能。全基因组方法和生物信息学预测已经产生了巨大的潜在增强子库，然而它们的时空活性通常仍有待于在体内验证。尽管最近在开发用于增强子评估的高通量策略方面取得了进展，但这些策略仍然主要限于无脊椎动物和体外细胞培养。在这里，我们设计了一个中等规模的方法来验证潜在的增强子在胚胎，鸡。使用不同报告载体的独特条形码，使我们能够通过一步RT-PCR检测单个胚胎中9个独立增强子的活性。该测定法足够灵敏以通过产生额外的条形码化载体来进一步扩大其能力。作为一种快速、灵敏和具有成本效益的方法来评估脊椎动物中的增强子活性，该方法为转基因动物的产生提供了一个重大的进步和一个有用的替代方案。Developmental Dynamics 244：1291 - 1299，2015.© 2015作者。出版社：Wiley Periodicals，Inc.代表美国解剖学家协会设计了一种新的策略，用于在鸡胚中快速验证增强子。用于快速克隆的简单载体的产生。许多增强子的活性可以使用基于PCR的策略在单个胚胎中检测到。该测定法足够灵敏以检测小部分细胞中的活性。
Enhancers are key elements to control gene expression in time and space and thus orchestrate gene function during development, homeostasis, and disease. Whole genome approaches and bioinformatic predictions have generated a tremendous pool of potential enhancers, however their spatiotemporal activity often remains to be validated in vivo. Despite recent progress in developing high throughput strategies for enhancer evaluation, these remain mainly restricted to invertebrates and in vitro cell culture. Here we design a medium‐scale method to validate potential enhancers in an amniote embryo, the chick. Using a unique barcode for different reporter vectors allows us to detect the activity of nine separate enhancers in a single embryo by one‐step RT‐PCR. The assay is sufficiently sensitive to expand its capacity further by generating additional barcoded vectors. As a rapid, sensitive, and cost‐effective way to assess enhancer activity in an amniote vertebrate, this method provides a major advance and a useful alternative to the generation of transgenic animals. Developmental Dynamics 244:1291–1299, 2015. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists Design of a new strategy for rapid enhancer validation in an amniote embryo, the chick. Generation of a simple vector for rapid cloning. The activity of many enhancers can be detected in a single embryo using a PCR‐based strategy. The assay is sufficiently sensitive to detect activity in a small fraction of cells.