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      Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreER T2 lines

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          Abstract

          The CreER T2/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreER T2 mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreER T2 mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreER T2 activity. While Ai14 and Ai3 easily recombine under basal CreER T2 activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.

          Electronic supplementary material

          The online version of this article (10.1007/s11248-019-00177-8) contains supplementary material, which is available to authorized users.

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          Most cited references 21

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          Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.

          Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17 beta-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.
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            Lineage tracing.

            Lineage tracing is the identification of all progeny of a single cell. Although its origins date back to developmental biology of invertebrates in the 19(th) century, lineage tracing is now an essential tool for studying stem cell properties in adult mammalian tissues. Lineage tracing provides a powerful means of understanding tissue development, homeostasis, and disease, especially when it is combined with experimental manipulation of signals regulating cell-fate decisions. Recently, the combination of inducible recombinases, multicolor reporter constructs, and live-cell imaging has provided unprecedented insights into stem cell biology. Here we discuss the different experimental strategies currently available for lineage tracing, their associated caveats, and new opportunities to integrate lineage tracing with the monitoring of intracellular signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.
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              The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage

              It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast1,2. A conflicting theory associates instead the first haematopoietic cells with a phenotypically differentiated endothelial cell with haematopoietic potential, i.e. a haemogenic endothelium3-5. Support for the haemangioblast concept was initially provided by the identification during embryonic stem (ES) cells differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components6,7. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo 8,9, the precise mechanism of generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating embryos and generates haematopoietic cells upon further culture. At the molecular level, we demonstrate that the transcription factor Scl/Tal110 is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1/AML111 is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge into a single linear developmental process the two a priori conflicting theories on the origin of haematopoietic development.
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                Author and article information

                Contributors
                konstantin.gaengel@igp.uu.se
                Journal
                Transgenic Res
                Transgenic Res
                Transgenic Research
                Springer International Publishing (Cham )
                0962-8819
                1573-9368
                22 October 2019
                22 October 2019
                2020
                : 29
                : 1
                : 53-68
                Affiliations
                [1 ]GRID grid.8993.b, ISNI 0000 0004 1936 9457, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, , Uppsala University, ; Dag Hammarskjöldsväg 20, 75185 Uppsala, Sweden
                [2 ]GRID grid.4714.6, ISNI 0000 0004 1937 0626, Integrated Cardio Metabolic Centre (ICMC), Department of Medicine Huddinge, , Karolinska Institutet, ; Novum, Blickagången 6, 141 57 Huddinge, Sweden
                Article
                177
                10.1007/s11248-019-00177-8
                7000517
                31641921
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100002794, Cancerfonden;
                Award ID: CAN2015/771
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Award ID: VR2015-00550
                Award ID: 542-2014-3535
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100010663, H2020 European Research Council;
                Award ID: 2011-294556
                Award ID: ERC-2014-CoG-646849
                Award Recipient :
                Funded by: Knut och Alice Wallenbergs Stiftelse (SE)
                Award ID: 2012.0272
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004063, Knut och Alice Wallenbergs Stiftelse;
                Award ID: 2015.0030
                Award Recipient :
                Funded by: Fondation Leducq (FR)
                Award ID: 14-CVD-02
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100014437, Wenner-Gren Stiftelserna;
                Categories
                Original Paper
                Custom metadata
                © Springer Nature Switzerland AG 2020

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