A medium-scale assay for enhancer validation in amniotes

作者：Chen Jingchen; Streit Andrea^*

来源：Developmental Dynamics, 2015, 244(10): 1291-1299.

DOI：10.1002/dvdy.24306

• conserved regulatory elements
• electroporation
• green fluorescent protein (GFP)
• otic placode
• PCR
• regulatory regions
• reporter plasmid

BackgroundEnhancers 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.
ResultsHere 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.
ConclusionsAs 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.

• 出版日期2015-10