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      AAV8 Ins1-Cre can produce efficient β-cell recombination but requires consideration of off-target effects

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          Abstract

          In vivo genetic manipulation is used to study the impact of gene deletion or re-expression on β-cell function and organism physiology. Cre-LoxP is a system wherein LoxP sites flanking a gene are recognized by Cre recombinase. Cre transgenic mice are the most prevalent technology used to deliver Cre but many models have caveats of off-target recombination, impaired β-cell function, and high cost of animal production. Inducible estrogen receptor conjugated Cre models face leaky recombination and confounding effects of tamoxifen. As an alternative, we characterize an adeno associated virus (AAV) with a rat insulin 1 promoter driving Cre recombinase (AAV8 Ins1-Cre) that is economical and rapid to implement, and has limited caveats. Intraperitoneal AAV8 Ins1-Cre produced efficient β-cell recombination, alongside some hepatic, exocrine pancreas, α-cell, δ-cell, and hypothalamic recombination. Delivery of lower doses via the pancreatic duct retained good rates of β-cell recombination and limited rates of off-target recombination. Unlike inducible Cre in transgenic mice, AAV8 Ins1-Cre required no tamoxifen and premature recombination was avoided. We demonstrate the utility of this technology by inducing hyperglycemia in inducible insulin knockout mice ( Ins1 −/−; Ins2 f/f). AAV-mediated expression of Cre in β-cells provides an effective alternative to transgenic approaches for inducible knockout studies.

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          A global double-fluorescent Cre reporter mouse.

          The Cre/loxP system has been used extensively for conditional mutagenesis in mice. Reporters of Cre activity are important for defining the spatial and temporal extent of Cre-mediated recombination. Here we describe mT/mG, a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision. We show that reporter expression is nearly ubiquitous, allowing visualization of fluorescent markers in live and fixed samples of all tissues examined. We further demonstrate that mG labeling is Cre-dependent, complementary to mT at single cell resolution, and distinguishable by fluorescence-activated cell sorting. Both membrane-targeted markers outline cell morphology, highlight membrane structures, and permit visualization of fine cellular processes. In addition to serving as a global Cre reporter, the mT/mG mouse may also be used as a tool for lineage tracing, transplantation studies, and analysis of cell morphology in vivo.
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            Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.

            Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics. Copyright © 2010 Elsevier Inc. All rights reserved.
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              The Dip Test of Unimodality

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                Author and article information

                Contributors
                tim.kieffer@ubc.ca
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                29 June 2020
                29 June 2020
                2020
                : 10
                Affiliations
                [1 ]ISNI 0000 0001 2288 9830, GRID grid.17091.3e, Department of Cellular and Physiological Sciences, , Life Sciences Institute, University of British Columbia, ; Vancouver, British Columbia Canada
                [2 ]ISNI 0000 0004 5930 4623, GRID grid.430579.c, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ; Madrid, Spain
                [3 ]Instituto de Investigación, Desarrollo e innovación en Biotecnología Sanitaria de Elche (IDiBE), Elche, Spain
                [4 ]ISNI 0000 0001 2288 9830, GRID grid.17091.3e, Department of Surgery, , University of British Columbia, ; Vancouver, British Columbia Canada
                [5 ]ISNI 0000 0001 2156 9982, GRID grid.266876.b, Northern Medical Program, University of Northern British Columbia, ; Prince George, British Columbia Canada
                [6 ]ISNI 0000 0001 0684 7788, GRID grid.414137.4, Department of Pathology and Laboratory Medicine, BC Children’s Hospital Research Institute, ; Vancouver, British Columbia Canada
                Article
                67136
                10.1038/s41598-020-67136-w
                7324556
                32601405
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef https://doi.org/10.13039/501100000024, Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada);
                Funded by: Vancouver Coastal Health (CIHR-UBC MD/PhD Studentship)
                Funded by: FundRef https://doi.org/10.13039/100009881, Juvenile Diabetes Research Foundation Canada (JDRF Canada);
                Funded by: FundRef https://doi.org/10.13039/100007476, Canadian Diabetes Association (Association Canadienne du Diabète);
                Categories
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                © The Author(s) 2020

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                gene delivery, genetic engineering

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