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      Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

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          Abstract

          Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER Tm and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1 PB - CreER Tm ; R26R lacZ mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.

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          Most cited references 79

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          Generalized lacZ expression with the ROSA26 Cre reporter strain.

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            Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.

            How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment.
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              Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.

              In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration. Copyright 2002 Elsevier Science (USA).
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                28 March 2012
                : 7
                : 3
                Affiliations
                [1 ]Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [2 ]Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [3 ]Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
                [4 ]Department of Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, United States of America
                University of Bremen, Germany
                Author notes

                Conceived and designed the experiments: RBR JK AAM MG MB ACP. Performed the experiments: RBR JK AAM GP MB. Analyzed the data: RBR JK AAM MG MB ACP. Contributed reagents/materials/analysis tools: MG. Wrote the paper: RBR JK MG MB ACP.

                Article
                PONE-D-11-23105
                10.1371/journal.pone.0033529
                3314663
                22470452
                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
                Page count
                Pages: 10
                Categories
                Research Article
                Biology
                Biotechnology
                Genetic Engineering
                Genetics
                Gene Expression
                Histology
                Model Organisms
                Animal Models
                Medicine
                Endocrinology
                Endocrine Physiology

                Uncategorized

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