A medium‐scale assay for enhancer validation in amniotes

Background 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. Results 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. Conclusions 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 Key Findings 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.