Optimization of reporter gene architecture for quantitative measurements of gene expression in the Drosophila embryo.

Quantitative assessment of gene regulation is critical for mathematical modeling of transcriptional systems for systems biology efforts. Enhancers, also termed cis-regulatory modules (CRMs), are the primary mediators of transcriptional regulation in higher eukaryotes; transcription factors binding to CRMs dictate the likelihood and frequency of promoter activation. To provide a suitable platform for in-depth CRM analysis, we adapted a targeted integration vector to compare action of basal promoters with diverse combination of TATA, Inr and DPE motifs, as well as a set of 3'-UTRs representative of those used in different reporter vectors. This "Honda" series of reporter gene vectors was activated by a regulatory element binding Dorsal and Twist activators suitable for transcription in the early Drosophila embryo. The diverse promoters functioned in a similar manner with minor quantitative differences, consistent with a lack of enhancer-promoter specificity. Constructs bearing SV40 3'-UTR sequences appeared to produce somewhat higher levels of mRNA. Confocal laser scanning microscopy revealed that the mRNA distribution produced by these constructs was punctate; this pattern appears to be dependent on 5'-UTR sequences, as an optimized vector including an alternate 5'-UTR produced a more even distribution, which may be preferable for quantitative imaging. This set of Honda vectors contains convenient sites for modification of basal promoter, 3' UTR, and enhancer, and will be useful for analysis of CRMs and quantitative studies of gene expression.