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Methods

In all of our projects we aim to obtain answers to our questions by combining molecular biology, biochemistry and high-resolution microscopy with genetic and phenotypic analyses at the level of the whole organism. We believe that this approach is much more informative than relying exclusively on tissue culture or in vitro systems.

Specifically, we use the following methods:

Molecular Biology: We generate numerous expression constructs for expression of a protein of interest in bacteria, in tissue culture and especially in transgenic flies. Most of these constructs are epitope tagged, often with tags suitable for life imaging, including GFP, RFP, Cherry etc. For the generation of these constructs we use the Gateway cloning system that allows the rapid generation of series of constructs with different N- and C-terminal tags.

More recently, we have started to use genomic BAC constructs, both for the introduction of mutations and for addition of epitope tags by recombineering in bacteria that allow the expression of the manipulated genes of interest under control of the endogenous regulatory sequences. These constructs are then used for the generation of transgenic fly lines using specific targeting sites in the genome. 

Biochemistry: We perform co-immunoprecipitation experiments from whole animal or tissue culture cell extracts to isolate protein complexes that are analyzed by immunoblots or by mass spectrometry. We also perform lipid-binding experiments with purified bacterially expressed fusion proteins. The function of kinases is analyzed by in vitro kinase assays.

Microscopy: All of our projects depend to a large extent on advanced light microscopy. The “work horse” for our laboratory is a confocal laser scanning microscope (Zeiss LSM 880 with Airyscan) that allows the simultaneous detection of multiple fluorescent dyes. With this setup we analyze both fixed specimens and perform life imaging of transgenic animals expressing fluorescent fusion proteins. We furthermore use a conventional fluorescence microscope (Zeiss AxioImager) equipped with structured illumination (Apotom) and a fully motorized X/Y/Z stage for automated simultaneous life recording of whole animals and cells. Our laboratory is also equipped with two conventional electron microscopes for the ultrastructural analysis of phenotypes and for the precise determination of the subcellular localization of proteins.

Genetics: In our laboratory all state of the art techniques for Drosophila genetics are established, including the generation of transgenic flies by target site specific germ line transformation, knock-out of genes by FLP-FRT-mediated recombination, overexpression of genes or RNAi constructs with the UAS-GAL4 system, clonal analysis by FLP-FRT-mediated recombination etc.