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Visualization and Identification of IL-7 Producing Cells in Reporter Mice

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      Abstract

      Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.

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      RAG-1-deficient mice have no mature B and T lymphocytes.

      The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.
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        Simple and highly efficient BAC recombineering using galK selection

        Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (λ) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive λ prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.
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          Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo.

          The naïve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of naïve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.
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            Author and article information

            Affiliations
            [1 ]Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institute of Health, Frederick, Maryland, United States of America
            [2 ]Mouse Cancer Genetics Program, National Cancer Institute, Frederick, Maryland, United States of America
            [3 ]Laboratory Animal Science Program (LASP), Science Applications International Corporation (SAIC), Cancer Research Center, National Cancer Institute, Frederick, Maryland, United States of America
            [4 ]Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
            [5 ]Laboratory of Experimental Immunology, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institute of Health, Frederick, Maryland, United States of America
            [6 ]The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
            [7 ]Department of Dermatology, Brigham and Women's Hospital, Boston, Massachusetts, United States of America
            [8 ]Human Retrovirus Section, Vaccine Branch, Center for Cancer Research NCI, Frederick, Maryland, United States of America
            [9 ]Gene Expression Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland, United States of America
            [10 ]Department of Immunology, University of Washington, Seattle, Washington, United States of America
            New York University School of Medicine, United States of America
            Author notes

            Conceived and designed the experiments: RIM AVW DWM RNG NAJ NGC SKD. Performed the experiments: RIM SW SML MI MA LF ACW DJS JAH MM JC DN LRS AGF KR. Analyzed the data: RIM WQL MRA AGF SKD. Contributed reagents/materials/analysis tools: DHDG BER. Wrote the paper: RIM SKD.

            [¤a]

            Current address: Laboratory of Gene Therapy and Primary Immunodeficiency, San Raffaele Telethon Institute for Gene Therapy, HSR-TIGET, Milano, Italy,

            [¤b]

            Current address: Genentech, Inc., Department of Molecular Biology, South San Francisco, California, United States of America

            [¤c]

            Current address: Laboratory of Biological Imaging, Immunology Frontier Research Center, Osaka University, Osaka, Japan

            [¤d]

            Current address: Twinbrook Flow Cytometry Facility, Lockheed Martin Contractor, RTB/NIAID/NIH, Rockville, Maryland, United States of America

            [¤e]

            Current address: Department of Biology, University of Puerto Rico Recinto Mayaguez, Mayaguez, Puerto Rico

            [¤f]

            Current address: Institute of Molecular and Cell Biology, Proteos, Singapore

            Contributors
            Role: Editor
            Journal
            PLoS One
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA )
            1932-6203
            2009
            10 November 2009
            : 4
            : 11
            2770321
            19907640
            09-PONE-RA-12143
            10.1371/journal.pone.0007637
            (Editor)
            This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
            Counts
            Pages: 14
            Categories
            Research Article
            Immunology
            Immunology/Leukocyte Development
            Immunology/Leukocyte Signaling and Gene Expression

            Uncategorized

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