In situ hybridization as a method to examine gene regulatory activity in vivo

Transcription factor-enhancer binding events are among the most well-studied protein-DNA interactions, allowing researchers to determine mechanisms of transcriptional activation or repression during development. While large-scale ChIP-sequence datasets, together with computational predictions and chromatin accessibility data, yield information on potential transcription factor binding activities, reporter gene assays provide measurable information on whether these binding activities are functional in particular cell types during development. Here, we present a detailed protocol to examine enhancer activity in Drosophila embryos using cloning, transgenesis, and in situ hybridization.