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      A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice

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          Abstract

          Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr− (H+/m−) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (I sc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔI sc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

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          Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.

          The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.
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            An animal model for cystic fibrosis made by gene targeting.

            Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.
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              A comparison of survival, growth, and pulmonary function in patients with cystic fibrosis in Boston and Toronto

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                Author and article information

                Contributors
                clarkel@missouri.edu
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                14 August 2019
                14 August 2019
                2019
                : 9
                : 11828
                Affiliations
                [1 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Dalton Cardiovascular Research Center, , University of Missouri, ; 134 Research Park Dr, Columbia, Missouri 65211-3300 USA
                [2 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Department of Biomedical Sciences, , University of Missouri, ; E102 Veterinary Medicine Bldg., Columbia, Missouri 65211 USA
                [3 ]ISNI 0000 0001 2164 3847, GRID grid.67105.35, Departments of Pediatrics, , Case Western Reserve University School of Medicine, ; Cleveland, Ohio USA
                [4 ]ISNI 0000 0001 2164 3847, GRID grid.67105.35, Departments of Physiology and Biophysics, , Case Western Reserve University School of Medicine, ; Cleveland, Ohio USA
                [5 ]ISNI 0000 0001 2164 3847, GRID grid.67105.35, Departments of Genetics and Genome Sciences, , Case Western Reserve University School of Medicine, ; Cleveland, Ohio USA
                [6 ]ISNI 0000 0001 2299 3507, GRID grid.16753.36, Human Molecular Genetics Program, Lurie Children’s Research Center, , Northwestern University Feinberg School of Medicine, ; Chicago, IL 60614 USA
                [7 ]ISNI 0000 0001 2155 0800, GRID grid.5216.0, Department of Histology and Embryology, School of Medicine, , University of Athens, ; Athens, Greece
                [8 ]InterRayBio, LLC, Baltimore, MD USA
                [9 ]ISNI 0000 0001 0703 675X, GRID grid.430503.1, Present Address: RNA Bioscience Initiative, , University of Colorado School of Medicine, ; Aurora, CO USA
                Author information
                http://orcid.org/0000-0002-4938-6261
                Article
                48105
                10.1038/s41598-019-48105-4
                6694137
                31413336
                3fe845bf-389c-44b5-bb04-f77796c5ce2c
                © The Author(s) 2019

                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/.

                History
                : 14 May 2018
                : 30 July 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000897, Cystic Fibrosis Foundation (CF Foundation);
                Award ID: HARRIS11G0
                Award ID: HARRIS14P0
                Award ID: CLARKE12G0
                Award ID: CLARKE14G0
                Award ID: CLARKE17G0
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000050, U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI);
                Award ID: HL094585
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100006501, Cystic Fibrosis Foundation Therapeutics (Cystic Fibrosis Foundation Therapeutics, Inc.);
                Award ID: CLARKE15XX0
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000062, U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases);
                Award ID: DK048816
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2019

                Uncategorized
                cystic fibrosis,gastrointestinal models,experimental models of disease
                Uncategorized
                cystic fibrosis, gastrointestinal models, experimental models of disease

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