An ideal transgenic gene expression system is inducible, non-leaky, and well tolerated by the target organism. While the former has been satisfactorily realized, leakiness and heavy physiological burden imposed by the existing systems are still prominent hurdles in their successful implementation. Here we describe a new system for non-leaky expression of transgenes in Drosophila. PRExpress is based on a single transgenic construct built from endogenous components, the inducible hsp70 promoter and a multimerized copy of a Polycomb response element (PRE) controlled by epigenetic chromatin regulators of the Polycomb group. We show that this system is non-leaky, rapidly and strongly inducible, and reversible. To make the application of PRExpress user-friendly, we deliver the construct via site-specific integration.

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.

Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation is bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. Here, we provide evidence for the existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, we observed a strong reporter inducibility in the embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in the embryo. Using re-chromatin immunoprecipitation experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes.

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