A conditional tissue-specific transgene expression system using inducible GAL4

Abstract

In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissuespecific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or ''larval bathing.'' Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre-and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

The ubiquitin–proteasome system postsynaptically regulates glutamatergic synaptic function

“…To determine the best timing interval, RU486 induction of expression of a reporter protein, τ tubulin-GFP, was examined by crossing the GeneSwitch MHC-GAL4 line with a UAS τ-tubulin GFP line. No GFP expression was detected in muscle cells up through the early third instar larval period (96 hours after egg laying), although a low level of GFP expression was observed in muscles of -RU486 control late wandering third instar larvae (data not shown), similar to a previous report (Osterwalder et al, 2001). Thus, early third instar larvae were used in subsequent experiments.…”

“…To generate heterozygote controls, UAS-DTS 5,7 and MHC-GAL4 lines were crossed with the wild-type Oregon-R (+/+) line. For inducible UAS transgene expression, the GeneSwitch system was utilized (Osterwalder et al, 2001). GeneSwitch uses a RU486-dependent GAL4-progesterone receptor fusion protein and the MHC-GeneSwitch GAL4 stock, generously provided by Dr. Haig Keshishian (Yale University).…”

“…To begin to assess this, we used the GeneSwitch system (Osterwalder et al, 2001) to attain temporal control over expression of the DTS proteasome mutant subunits. The GeneSwitch MHC-GAL4 line was crossed with the UAS DTS proteasome mutant line.…”

Comparative transcriptomics across 14 Drosophila species reveals signatures of longevity

“…In this resource, the transgenic shRNA is placed under UAS/GAL4 promoter and can be induced by feeding flies with the drug RU486 (Osterwalder, Yoon, White & Keshishian, 2001). To test if manipulation of individual genes identified as a part of the longevity signatures affects lifespan, we examined 20 lines representing five genes with positive correlation to longevity and seven genes with negative correlation to longevity in both male and female transgenic lines (Figure 4, Table S4).…”

Morphometric Analysis of Huntington’s Disease Neurodegeneration in Drosophila

“…The recombinase can then remove a terminator cassette fl anked by FLP recognition target sequences that is placed in front of GAL4, thus allowing expression of GAL4 and activation of the GAL4-UAS controlled transgene [ 27,29,30 ] . Other inducible drivers are controlled by speci fi c ligands such as mifepristone/ RU486 in a dose-dependent manner [ 27,31,32 ] . Where expression levels are critical, it is important to verify the relative levels in induced vs. uninduced systems.…”