A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells.

Autor: Tichy ED; Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA., Sidibe DK; Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA., Greer CD; Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA.; Cell and Molecular Biology Graduate Program, The University of Pennsylvania, Philadelphia, PA, USA., Oyster NM; Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA., Rompolas P; Cell and Molecular Biology Graduate Program, The University of Pennsylvania, Philadelphia, PA, USA.; Department of Dermatology, Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA., Rosenthal NA; The Jackson Laboratory, Bar Harbor, ME, USA.; Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA.; The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.; Australian Regenerative Medicine Institute, Monash University, Melbourne, VIC, Australia.; National Heart and Lung Institute, Imperial College London, London, UK., Blau HM; Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford School of Medicine, Stanford, CA, USA., Mourkioti F; Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA. fmour@pennmedicine.upenn.edu.; Cell and Molecular Biology Graduate Program, The University of Pennsylvania, Philadelphia, PA, USA. fmour@pennmedicine.upenn.edu.; Department of Cell and Developmental Biology, Penn Institute of Regenerative Medicine, Musculoskeletal Regeneration Program, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA. fmour@pennmedicine.upenn.edu.; Musculoskeletal Regeneration Program, Department of Orthopaedic Surgery and Cell and Developmental Biology, Penn Institute of Regenerative Medicine, The University of Pennsylvania, 3450 Hamilton Walk, 112A Stemmler Hall, Philadelphia, PA, 19104-6081, USA. fmour@pennmedicine.upenn.edu.
Jazyk: angličtina
Zdroj: Skeletal muscle [Skelet Muscle] 2018 Aug 24; Vol. 8 (1), pp. 27. Date of Electronic Publication: 2018 Aug 24.
DOI: 10.1186/s13395-018-0169-7
Abstrakt: Background: Pax7 is a transcription factor involved in the specification and maintenance of muscle stem cells (MuSCs). Upon injury, MuSCs leave their quiescent state, downregulate Pax7 and differentiate, contributing to skeletal muscle regeneration. In the majority of regeneration studies, MuSCs are isolated by fluorescence-activated sorting (FACS), based on cell surface markers. It is known that MuSCs are a heterogeneous population and only a small percentage of isolated cells are true stem cells that are able to self-renew. A strong Pax7 reporter line would be valuable to study the in vivo behavior of Pax7-expressing stem cells.
Methods: We generated and characterized the muscle properties of a new transgenic Pax7EGFP mouse. Utilizing traditional immunofluorescence assays, we analyzed whole embryos and muscle sections by fluorescence microscopy, in addition to whole skeletal muscles by 2-photon microscopy, to detect the specificity of EGFP expression. Skeletal muscles from Pax7EGFP mice were also evaluated in steady state and under injury conditions. Finally, MuSCs-derived from Pax7EGFP and control mice were sorted and analyzed by FACS and their myogenic activity was comparatively examined.
Results: Our studies provide a new Pax7 reporter line with robust EGFP expression, detectable by both flow cytometry and fluorescence microscopy. Pax7EGFP-derived MuSCs have identical properties to that of wild-type MuSCs, both in vitro and in vivo, excluding any positional effect due to the transgene insertion. Furthermore, we demonstrated high specificity of EGFP to label MuSCs in a temporal manner that recapitulates the reported Pax7 expression pattern. Interestingly, immunofluorescence analysis showed that the robust expression of EGFP marks cells in the satellite cell position of adult muscles in fixed and live tissues.
Conclusions: This mouse could be an invaluable tool for the study of a variety of questions related to MuSC biology, including but not limited to population heterogeneity, polarity, aging, regeneration, and motility, either by itself or in combination with mice harboring additional genetic alterations.
Databáze: MEDLINE
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