Massively Parallel Reporter Assays for High-Throughput In Vivo Analysis of Cis-Regulatory Elements.
Massively Parallel Reporter Assays for High-Throughput In Vivo Analysis of Cis-Regulatory Elements.
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DOI：
10.3390/jcdd10040144
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发表时间：
2023-03-29
影响因子：
2.4
通讯作者：
中科院分区：
文献类型：
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描述性基因组学技术的快速发展，推动了心血管基因表达和表型之间假设联系的急剧增加。然而，这些假设的体内测试主要被降级为缓慢，昂贵和线性生成的转基因小鼠。在基因组顺式调控元件的研究中，产生具有转基因报告基因或顺式调控元件敲除的小鼠仍然是标准方法。虽然获得的数据质量很高，但这种方法不足以跟上候选人的确定，因此在选择候选人进行验证时会产生偏差。然而，跨一系列学科的最新进展正在融合，以实现可以以高通量方式进行的功能性基因组测定。在这里，我们回顾了这样一种方法，大规模并行报告基因检测（MPRAs），其中成千上万的候选基因组调控元件的活动，同时通过下一代测序的条形码报告转录本进行评估。我们讨论了MPRA设计和使用的最佳实践，重点是实际考虑，并回顾了这种新兴技术是如何成功地在体内部署的。最后，我们讨论了如何MPRA可能会演变，并在未来的心血管研究中使用。
The rapid improvement of descriptive genomic technologies has fueled a dramatic increase in hypothesized connections between cardiovascular gene expression and phenotypes. However, in vivo testing of these hypotheses has predominantly been relegated to slow, expensive, and linear generation of genetically modified mice. In the study of genomic cis-regulatory elements, generation of mice featuring transgenic reporters or cis-regulatory element knockout remains the standard approach. While the data obtained is of high quality, the approach is insufficient to keep pace with candidate identification and therefore results in biases introduced during the selection of candidates for validation. However, recent advances across a range of disciplines are converging to enable functional genomic assays that can be conducted in a high-throughput manner. Here, we review one such method, massively parallel reporter assays (MPRAs), in which the activities of thousands of candidate genomic regulatory elements are simultaneously assessed via the next-generation sequencing of a barcoded reporter transcript. We discuss best practices for MPRA design and use, with a focus on practical considerations, and review how this emerging technology has been successfully deployed in vivo. Finally, we discuss how MPRAs are likely to evolve and be used in future cardiovascular research.
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Emmer BT;Sherman EJ;Lascuna PJ;Graham SE;Willer CJ;Ginsburg D
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Ginsburg D
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