title: A medium-scale assay for enhancer validation in amniotes.
abstract: 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.
authors.affiliation: Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London, London, United Kingdom.
authors.forename: Jingchen
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Craniofacial Development and Stem Cell Biology, Dental Institute, King's College London, London, United Kingdom.
authors.forename: Andrea
authors.initials: A
authors.lastname: Streit
mesh_terms: D000818:Animals; D002642:Chick Embryo; D058893:DNA Barcoding, Taxonomic; D015509:Developmental Biology / Q000379:methods*; D018274:Electroporation; D004742:Enhancer Elements, Genetic; D005822:Genetic Vectors
keywords: PCR; conserved regulatory elements; electroporation; green fluorescent protein (GFP); otic placode; regulatory regions; reporter plasmid