title: A Noninvasive In Vitro Monitoring System Reporting Skeletal Muscle Differentiation.
abstract: Monitoring of cell differentiation is a crucial aspect of cell-based therapeutic strategies depending on tissue maturation. In this study, we have developed a noninvasive reporter system to trace murine skeletal muscle differentiation. Either a secreted bioluminescent reporter (Metridia luciferase) or a fluorescent reporter (green fluorescent protein [GFP]) was placed under the control of the truncated muscle creatine kinase (MCK) basal promoter enhanced by variable numbers of upstream MCK E-boxes. The engineered pE3MCK vector, coding a triple tandem of E-Boxes and the truncated MCK promoter, showed twentyfold higher levels of luciferase activation compared with a Cytomegalovirus (CMV) promoter. This newly developed reporter system allowed noninvasive monitoring of myogenic differentiation in a straining bioreactor. Additionally, binding sequences of endogenous microRNAs (miRNAs; seed sequences) that are known to be downregulated in myogenesis were ligated as complementary seed sequences into the reporter vector to reduce nonspecific signal background. The insertion of seed sequences improved the signal-to-noise ratio up to 25% compared with pE3MCK. Due to the highly specific, fast, and convenient expression analysis for cells undergoing myogenic differentiation, this reporter system provides a powerful tool for application in skeletal muscle tissue engineering.
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Deniz
authors.initials: D
authors.lastname: Öztürk-Kaloglu
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: David
authors.initials: D
authors.lastname: Hercher
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Philipp
authors.initials: P
authors.lastname: Heher
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Katja
authors.initials: K
authors.lastname: Posa-Markaryan
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Simon
authors.initials: S
authors.lastname: Sperger
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Alice
authors.initials: A
authors.lastname: Zimmermann
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Susanne
authors.initials: S
authors.lastname: Wolbank
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Heinz
authors.initials: H
authors.lastname: Redl
authors.affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology , Vienna, Austria .
authors.forename: Ara
authors.initials: A
authors.lastname: Hacobian
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D024510:Muscle Development / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D059629:Signal-To-Noise Ratio
chemical_list: D049452:Green Fluorescent Proteins; D003402:Creatine Kinase
keywords: cell differentiation; monitoring myogenesis; reporter systems; skeletal muscle