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    Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation

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    Author
    Winbanks, CE; Beyer, C; Qian, H; Gregorevic, P
    Date
    2012-12-12
    Source Title
    PLOS ONE
    Publisher
    PUBLIC LIBRARY SCIENCE
    University of Melbourne Author/s
    Gregorevic, Paul; Qian, Hongwei
    Affiliation
    Physiology
    Metadata
    Show full item record
    Document Type
    Journal Article
    Citations
    Winbanks, C. E., Beyer, C., Qian, H. & Gregorevic, P. (2012). Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation. PLOS ONE, 7 (12), https://doi.org/10.1371/journal.pone.0051627.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/240783
    DOI
    10.1371/journal.pone.0051627
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520959
    Abstract
    Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

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