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Ludwig-Maximilians-University Munich

6 ARTICLES PUBLISHED IN JoVE

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Biology

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Bernd Rädle *1, Andrzej J. Rutkowski *2, Zsolt Ruzsics 1, Caroline C. Friedel 3, Ulrich H. Koszinowski 1, Lars Dölken 2
1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

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Behavior

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila
Sara Batelli 1, Malte Kremer 1,2, Christophe Jung 1, Ulrike Gaul 1
1Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, 2Janelia Farm Research Campus, Howard Hughes Medical Institute

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

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Genetics

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation
Kathrin Davari *1, Johannes Lichti *1, Caroline C. Friedel 2, Elke Glasmacher 1,3
1Institute for Diabetes and Obesity (IDO), German Center for Diabetes Research (DZD), Helmholtz Zentrum München, 2Institute for Informatics, Ludwig-Maximilians-Universität München, 3Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

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Biochemistry

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Christophe Jung 1, Max Schnepf 1, Peter Bandilla 1, Ulrich Unnerstall 1, Ulrike Gaul 1
1Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

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Genetics

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches
Shao-Yen Kao *1, Elena Nikonova *1, Keshika Ravichandran *1, Maria L. Spletter 1,2
1Biomedical Center, Department of Physiological Chemistry, Ludwig-Maximilians-University Munich, 2Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Ludwig-Maximilians-University Munich

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

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Biology

An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers
Shuxia Fan 1,2, Ben Raude 1,2,3, Florian Gaertner 1,2,4,5
1Department of Medicine I, University Hospital, LMU Munich, 2Institute of Surgical Research at the Walter Brendel Centre of Experimental Medicine, University Hospital, LMU Munich, 3Department of Vascular Surgery, Charité-Universitätsmedizin Berlin, Freie Universität Berlin and Humboldt-Universität zu Berlin, 4Interfaculty Center for Endocrine and Cardiovascular Disease Network Modelling and Clinical Transfer, Ludwig-Maximilians-University Munich, 5DZHK (German Centre for Cardiovascular Research), Partner site Munich Heart Alliance

A detailed protocol for imaging single migrating platelets using RGD-functionalized avidin-biotin tethers with tunable density is provided, revealing that platelets generate enough force to rupture the avidin-biotin bond.

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