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German Cancer Research Center (DKFZ)

9 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems
Nazim El-Andaloussi *1,2, Barbara Leuchs *1, Serena Bonifati 1,2, Jean Rommelaere 1,2, Antonio Marchini 1,2
1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ)

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

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Neuroscience

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation
Wilson Pak-Kin Lou 1, Avni Baser 1, Stefan Klußmann 1, Ana Martin-Villalba 1
1Department of Molecular Neurobiology, German Cancer Research Center (DKFZ)

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

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Developmental Biology

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression
Sheena Pinto *1, Hans-Jürgen Stark *2, Iris Martin 2, Petra Boukamp 2, Bruno Kyewski 1
1Division of Developmental Immunology, German Cancer Research Center (DKFZ), 2Genetics of Skin Carcinogenesis, German Cancer Research Center (DKFZ)

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

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Cancer Research

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer
Susan Kochall 1, May-Linn Thepkaysone 1, Sebastián A. García 1, Alexander M. Betzler 1, Jürgen Weitz 1,2,3, Christoph Reissfelder 1, Sebastian Schölch 1,2,3
1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2German Cancer Consortium (DKTK), 3German Cancer Research Center (DKFZ)

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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Cancer Research

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer
Alexander M. Betzler 1, Susan Kochall 1, Linda Blickensdörfer 2, Sebastian A. Garcia 1, May-Linn Thepkaysone 1, Lahiri K. Nanduri 1, Michael H. Muders 3, Jürgen Weitz 1,4,5, Christoph Reissfelder 1, Sebastian Schölch 1,4,5
1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2Department of General, Gastrointestinal and Transplant Surgery, University of Heidelberg, 3Department of Pathology, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 4German Cancer Consortium (DKTK), 5German Cancer Research Center (DKFZ)

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

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Genetics

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes
Wilson Pak-Kin Lou *1,2, Liqun Wang *3, Cheng Long 1, Lei Liu 3, Ji-Feng Fei 3
1School of Life Sciences, South China Normal University, 2The Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 3Institute for Brain Research and Rehabilitation (IBRR), South China Normal University

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

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Genetics

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids
Anne-Marlen Gaebler 1, Alexander Hennig 1, Katharina Buczolich 1, Jürgen Weitz 1, Thilo Welsch 1, Daniel E. Stange 1, Kristin Pape 1
1Department of Visceral-, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Technische Universität Dresden

This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

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Cancer Research

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq
Ashwin Narayanan 1, Enrique Blanco-Carmona 2, Engin Demirdizen 1, Xueyuan Sun 1,3, Christel Herold-Mende 4, Matthias Schlesner 2, Sevin Turcan 1
1Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, 2Bioinformatics and Omics Data Analytics, German Cancer Research Center (DKFZ), 3Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), 4Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, INF 400, Heidelberg, Germany

Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies.

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Immunology and Infection

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions
Megan L. Stanifer 1,2, Steeve Boulant 2,3,4
1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, 3Department of Infectious Diseases, Virology, Heidelberg University Hospital, 4Research Group “Cellular Polarity and Viral Infection”, German Cancer Research Center (DKFZ)

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

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