title: Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.
abstract: Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail.
authors: Institute for Cell and Molecular Biosciences, Campus for Ageing and Vitality, University of Newcastle, Newcastle-upon-Tyne, NE4 5PL, United Kingdom.
authors: Filippo
authors: F
authors: Scialo
authors: Institute for Cell and Molecular Biosciences, Campus for Ageing and Vitality, University of Newcastle, Newcastle-upon-Tyne, NE4 5PL, United Kingdom.
authors: Ashwin
authors: A
authors: Sriram
authors: Institute for Cell and Molecular Biosciences, Campus for Ageing and Vitality, University of Newcastle, Newcastle-upon-Tyne, NE4 5PL, United Kingdom.
authors: Rhoda
authors: R
authors: Stefanatos
authors: Institute for Cell and Molecular Biosciences, Campus for Ageing and Vitality, University of Newcastle, Newcastle-upon-Tyne, NE4 5PL, United Kingdom.
authors: Alberto
authors: A
authors: Sanz
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D015735:Mifepristone / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015735:Mifepristone