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German Center for Infection Research (DZIF)

6 ARTICLES PUBLISHED IN JoVE

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Biology

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Joachim Hauber 1
1Department of Cell Biology and Virology, Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

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Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
Eike Kienle *1, Elena Senís *1, Kathleen Börner 2, Dominik Niopek 1, Ellen Wiedtke 1, Stefanie Grosse 1, Dirk Grimm 1
1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

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Immunology and Infection

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Thomas Tu 1,2, Stephan Urban 1,3
1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Gastroenterology and Liver Research Laboratory, Ingham Institute, 3German Center for Infection Research (DZIF)

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1–2 copies) integration using inverse nested PCR.

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Immunology and Infection

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites
Julia K. Bialek *1,2, Thomas Walther *1, Joachim Hauber 1,3, Ulrike C. Lange 1,2,3
1Heinrich Pette Institute, Leibniz Institute for Experimental Virology, 2Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, 3German Center for Infection Research (DZIF)

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

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Immunology and Infection

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Marc Herb 1, Alina Farid 1, Alexander Gluschko 1, Martin Krönke 1,2,3,4, Michael Schramm 1
1Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, 2Center for Molecular Medicine Cologne, 3Cologne Cluster of Excellence on Cellular Stress Responses in Aging-associated Diseases (CECAD), 4German Center for Infection Research (DZIF)

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

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Bioengineering

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants
Kleopatra Rapti 1,2, Olena Maiakovska 1,2, Jonas Becker 1,2, Joanna Szumska 1,2, Margarita Zayas 1,2, Felix Bubeck 1,2, Jixin Liu 1,2, Emma Gerstmann 1,2, Chiara Krämer 1,2, Ellen Wiedtke 1,2, Dirk Grimm 1,2,3,4,5
1Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, 2BioQuant Center, BQ0030, University of Heidelberg, 3Cluster of Excellence CellNetworks, 4German Center for Infection Research (DZIF), 5German Center for Cardiovascular Research (DZHK)

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

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