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Max Planck Institute of Molecular Cell Biology and Genetics

11 ARTICLES PUBLISHED IN JoVE

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Biology

Molecular Evolution of the Tre Recombinase
Frank Buchholz 1
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.

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Biology

MISSION esiRNA for RNAi Screening in Mammalian Cells
Mirko Theis 1, Frank Buchholz 1
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.

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Biology

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
Alexis B. Webb 1, Daniele Soroldoni 1, Annelie Oswald 1, Johannes Schindelin 1, Andrew C. Oates 1
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.

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Biology

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus
Mark Slabodnick 1,2, Bram Prevo 1,3, Peter Gross 1,4, Janet Sheung 1,5, Wallace Marshall 1,2
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.

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Biology

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish
Michael Weber 1, Michaela Mickoleit 1, Jan Huisken 1
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.

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Developmental Biology

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development
Jaroslav Icha *1, Christopher Schmied *1, Jaydeep Sidhaye 1, Pavel Tomancak 1, Stephan Preibisch 1,2,3, Caren Norden 1
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.

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Developmental Biology

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers
Ivana Viktorinová 1,2, Robert Haase *1,2, Tobias Pietzsch *1,2, Ian Henry 1,2, Pavel Tomancak 1,2
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.

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Neuroscience

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation
Nereo Kalebic 1,2, Barbara Langen 1,3, Jussi Helppi 1, Hiroshi Kawasaki 4, Wieland B. Huttner 1
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.

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Bioengineering

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection
Gabriella Shull *1,2, Christiane Haffner *3, Wieland B. Huttner 3, Elena Taverna 3,4, Suhasa B. Kodandaramaiah 1,5,6
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.

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Biology

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos
Anjalie Schlaeppi 1, Alyssa Graves 1, Michael Weber 1, Jan Huisken 1
1Morgridge Institute for Research, Madison, WI

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

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Developmental Biology

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification
Lidiia Tynianskaia 1, Nesil Eşiyok 1, Wieland B. Huttner 2, Michael Heide 1,2
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.

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