CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.

Autor: Kaltenbacher T; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Löprich J; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Maresch R; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Weber J; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Müller S; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Oellinger R; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Groß N; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Griger J; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., de Andrade Krätzig N; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Avramopoulos P; Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany.; DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany., Ramanujam D; Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany.; DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany., Brummer S; Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany., Widholz SA; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Bärthel S; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.; Institute of Experimental Cancer Therapy, Technical University of Munich, Munich, Germany., Falcomatà C; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.; Institute of Experimental Cancer Therapy, Technical University of Munich, Munich, Germany., Pfaus A; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany., Alnatsha A; Department of Medicine II, University Hospital, LMU Munich, Munich, Germany., Mayerle J; Department of Medicine II, University Hospital, LMU Munich, Munich, Germany.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany., Schmidt-Supprian M; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.; Institute of Experimental Hematology, School of Medicine, Technical University of Munich, Munich, Germany., Reichert M; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany., Schneider G; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany., Ehmer U; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany., Braun CJ; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany.; Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich, Germany.; Hopp Children's Cancer Center Heidelberg (KiTZ), German Cancer Research Center (DKFZ), Heidelberg, Germany., Saur D; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany.; Institute of Experimental Cancer Therapy, Technical University of Munich, Munich, Germany.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany., Engelhardt S; Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany.; DZHK (German Centre for Cardiovascular Research), Partner Site Munich Heart Alliance, Munich, Germany., Rad R; Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.; Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. roland.rad@tum.de.; Department of Medicine II, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, Munich, Germany. roland.rad@tum.de.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2022 Apr; Vol. 17 (4), pp. 1142-1188. Date of Electronic Publication: 2022 Mar 14.
DOI: 10.1038/s41596-021-00677-0
Abstrakt: Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.
(© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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