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      Generating mouse models for biomedical research: technological advances

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          ABSTRACT

          Over the past decade, new methods and procedures have been developed to generate genetically engineered mouse models of human disease. This At a Glance article highlights several recent technical advances in mouse genome manipulation that have transformed our ability to manipulate and study gene expression in the mouse. We discuss how conventional gene targeting by homologous recombination in embryonic stem cells has given way to more refined methods that enable allele-specific manipulation in zygotes. We also highlight advances in the use of programmable endonucleases that have greatly increased the feasibility and ease of editing the mouse genome. Together, these and other technologies provide researchers with the molecular tools to functionally annotate the mouse genome with greater fidelity and specificity, as well as to generate new mouse models using faster, simpler and less costly techniques.

          Abstract

          Summary: Newer molecular technologies to precisely and efficiently manipulate the mammalian genome are enabling the production of more scientifically valuable animal models.

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          Most cited references106

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          Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

          Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.
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            Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.

            An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.
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              Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.

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                Author and article information

                Journal
                Dis Model Mech
                Dis Model Mech
                DMM
                dmm
                Disease Models & Mechanisms
                The Company of Biologists Ltd
                1754-8403
                1754-8411
                1 January 2019
                8 January 2019
                8 January 2019
                : 12
                : 1
                : dmm029462
                Affiliations
                [1 ]Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center , Omaha, NE 68106-5915, USA
                [2 ]Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center , Omaha, NE 68106-5915, USA
                [3 ]Department of Surgery, School of Medicine, University of California , Davis, CA 95618, USA
                [4 ]Mouse Biology Program, University of California , Davis, CA 95618, USA
                Author notes
                [* ]Author for correspondence ( kclloyd@ 123456ucdavis.edu )
                Author information
                http://orcid.org/0000-0002-8022-4033
                http://orcid.org/0000-0002-5318-4144
                Article
                DMM029462
                10.1242/dmm.029462
                6361157
                30626588
                ed0660c2-ea8e-47a4-9830-808bbcf3f562
                © 2019. Published by The Company of Biologists Ltd

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                History
                Categories
                Mouse Models
                At A Glance

                Molecular medicine
                crispr,genome editing,mouse,mutagenesis
                Molecular medicine
                crispr, genome editing, mouse, mutagenesis

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