In a recent issue of BioTechniques, Voutev et al. (2016) describe a fast, efficient, and effective method for the in vivo identification of transcriptional enhancers using transgenic reporter genes.
Enhancer discovery is a critical aspect of functional genomics studies that seek to elucidate the role of noncoding sequences in regulating gene expression. While genome-scale enhancer detection using high-throughput next generation sequencing-based methods is now feasible in model organisms such as Drosophila melanogaster, these methods are as yet not tractable to studying a large number of non-model or emerging model insects. Furthermore, these methods cannot be used to screen genomic regions for patterned regulatory activity during development.
The solution adopted by Voutev et al. uses ~20 kb sized clones from the CHORI-322 genomic libraries that cover the entire Drosophila genome (created by Venken et al., 2009) and contain both a screenable marker (mini-white+) and a phiC31 attB site to create reporter constructs for testing in transgenic Drosophila.
The elegance of this method lies in the creation of reporter constructs by simply linearizing each clone of interest using a clone-specific unique restriction enzyme. Then a reporter cassette consisting of a synthetic minimal promoter driving tdTomato expression and a 3′ SV40 terminator PCR product can be introduced using Gibson assembly PCR without the need for any intermediate cloning steps.
As a proof of concept, three lines of transgenic flies carrying reporter constructs covering ~60 kb of the D. melanogaster teashirt (tsh) were analyzed for expression in larval imaginal discs, whole embryos, and the larval nervous system with each of them driving reporter gene expression in one or more developmental stages thus identifying multiple regulatory regions.
The attractiveness of this technique lies in its easy applicability to a host of different nonmodel insects with sequenced genomes.
However, a limitation of this approach is the requirement of genomic libraries in any sequenced species in order to test regions of interest for regulatory activity. Furthermore, the sizes of these libraries need to be relatively small (~20 kb) for efficient integration into the genome using the phiC31 integrase system.
The investment required to generate such libraries is estimably large and could pose a signficant hurdle for use of such an application.
Another caveat is that positive clones need to be further characterized to identify the minimal enhancer fragment(s).
This method also does not inform us of the presence of multiple enhancers in a clone that is being tested.
Nevertheless, for those species where this method is feasible, complementing this assay with computational enhancer discovery methods and/or epigenetic/chromatin profiling methods to zero in on the minimal regulatory regions will greatly aid in the rapid identification and annotation of these critical components of the transcriptional machinery.
The power of this technique can be elevated by the maturation of transgenic technology in nonmodel insects allowing researchers to test in the native species rather than the widely accepted practice of testing in a heterologous context in D. melanogaster.