Max Planck Institute of Molecular Cell Biology and Genetics View Institution's Website 12 articles published in JoVE Developmental Biology Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification Lidiia Tynianskaia1, Nesil Eşiyok1, Wieland B. Huttner2, Michael Heide1,2 1German Primate Center, Leibniz Institute for Primate Research, 2Max Planck Institute of Molecular Cell Biology and Genetics The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling. Bioengineering Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection Gabriella Shull*1,2, Christiane Haffner*3, Wieland B. Huttner3, Elena Taverna3,4, Suhasa B. Kodandaramaiah1,5,6 1Department of Biomedical Engineering, University of Minnesota, 2Department of Biomedical Engineering, Duke University, 3Max Planck Institute of Molecular Cell Biology and Genetics, 4Max Planck Institute for Evolutionary Anthropology, 5Department of Mechanical Engineering, University of Minnesota, 6Graduate Program in Neuroscience, University of Minnesota This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution. Neuroscience In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation Nereo Kalebic1,2, Barbara Langen1,3, Jussi Helppi1, Hiroshi Kawasaki4, Wieland B. Huttner1 1Max Planck Institute of Molecular Cell Biology and Genetics, 2Human Technopole, 3Landesdirektion Sachsen, 4Department of Medical Neuroscience, Graduate School of Medical Sciences, Kanazawa University Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo. Developmental Biology Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers Ivana Viktorinová1,2, Robert Haase*1,2, Tobias Pietzsch*1,2, Ian Henry1,2, Pavel Tomancak1,2 1Max Planck Institute of Molecular Cell Biology and Genetics, 2Center for Systems Biology Dresden This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins. Biology Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging Liliana Malinovska1, Simon Alberti1 1Max-Planck Institute for Molecular Cell Biology and Genetics The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum. Developmental Biology Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development Jaroslav Icha*1, Christopher Schmied*1, Jaydeep Sidhaye1, Pavel Tomancak1, Stephan Preibisch1,2,3, Caren Norden1 1Max Planck Institute of Molecular Cell Biology and Genetics, 2HHMI Janelia Research Campus, 3Berlin Institute of Medical Systems Biology of the Max Delbrück Center Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets. Biology Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells Alexis B. Webb1, Daniele Soroldoni1, Annelie Oswald1, Johannes Schindelin1, Andrew C. Oates1 1Max Planck Institute of Molecular Cell Biology and Genetics Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro. Biology Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish Michael Weber1, Michaela Mickoleit1, Jan Huisken1 1Huisken Lab, Max Planck Institute of Molecular Cell Biology and Genetics The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose. Biology Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus Mark Slabodnick1,2, Bram Prevo1,3, Peter Gross1,4, Janet Sheung1,5, Wallace Marshall1,2 1Physiology Course, Marine Biological Laboratory, 2Department of Biochemistry & Biophysics, University of California San Francisco, 3Department of Physics and Astronomy, Vrije Universiteit Amsterdam, 4Max Planck Institute of Molecular Cell Biology and Genetics, 5Department of Physics, University of Illinois Urbana-Champaign The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells. By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding. Biology MISSION esiRNA for RNAi Screening in Mammalian Cells Mirko Theis1, Frank Buchholz1 1Max Planck Institute of Molecular Cell Biology and Genetics Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results. Biology Molecular Evolution of the Tre Recombinase Frank Buchholz1 1Max Plank Institute for Molecular Cell Biology and Genetics, Dresden Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine. Biology Principles of Site-Specific Recombinase (SSR) Technology Frank Bucholtz1 1Max Plank Institute for Molecular Cell Biology and Genetics, Dresden The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.