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German Research Center for Environmental Health

5 ARTICLES PUBLISHED IN JoVE

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Biochemistry

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins
Jenna K. Zalewski 1, Simone Heber 1,2, Joshua H. Mo 1, Keith O'Conor 1, Jeffrey D. Hildebrand 1, Andrew P. VanDemark 1
1Department of Biological Sciences, University of Pittsburgh, 2Institute of Structural Biology, German Research Center for Environmental Health

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

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Genetics

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
Anna Köferle 1, Stefan H. Stricker 1,2
1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian-Universität, BioMedical Center, 2Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

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Genetics

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)
Christopher T. Breunig 1,2, Andrea M. Neuner 1,2, Jessica Giehrl-Schwab 3, Wolfgang Wurst 3, Magdalena Götz 2,4, Stefan H. Stricker 1,2
1MCN Junior Research Group, Munich Center for Neurosciences, Ludwig Maximilian Universitat, BioMedical Center, 2Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, 3Institute of Developmental Genetics, Helmholtz Zentrum, German Research Center for Environmental Health, 4Physiological Genomics, Ludwig Maximilian Universitat, BioMedical Center

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

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Neuroscience

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis
Christian Friess 1, Magdalena Götz 1,2,3, Jacob Kjell 1,2,4
1Division of Physiological Genomics, Biomedical Center, Ludwig Maximilian University of Munich, 2Institute for Stem Cell Research, Helmholtz Zentrum München, 3SYNERGY, Excellence Cluster Systems Neurology, University of Munich, 4Department of Clinical Neuroscience, Karolinska Institutet

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

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Neuroscience

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes
Bob A. Hersbach 1,2,3, Tatiana Simon 2, Giacomo Masserdotti 1,2
1Institute of Stem Cell Research, Helmholtz Zentrum München, German Research Center for Environmental Health, 2Department of Physiological Genomics, Biomedical Center Munich, Ludwig-Maximilians University, 3Graduate School of Systemic Neurosciences, BioCenter, Ludwig-Maximilians University

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

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