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Limitations of nlsβ-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

1997; Wiley; Volume: 248; Issue: 1 Linguagem: Inglês

10.1002/(sici)1097-0185(199705)248

1097-0185

Jiwei Yang, Martin P. Ontell, Robert G. Kelly, Simon C. Watkins, Marcia Ontell,

RNA Research and Splicing

The Anatomical RecordVolume 248, Issue 1 p. 40-50 MyologyFree Access Limitations of nlsβ-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer Jiwei Yang, Jiwei Yang Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorMartin P. Ontell, Martin P. Ontell Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorRobert Kelly, Robert Kelly Department of Molecular Biology, Pasteur Institute, Paris, FranceSearch for more papers by this authorSimon C. Watkins, Simon C. Watkins Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorMarcia Ontell, Corresponding Author Marcia Ontell Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaDepartment of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261Search for more papers by this author Jiwei Yang, Jiwei Yang Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorMartin P. Ontell, Martin P. Ontell Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorRobert Kelly, Robert Kelly Department of Molecular Biology, Pasteur Institute, Paris, FranceSearch for more papers by this authorSimon C. Watkins, Simon C. Watkins Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaSearch for more papers by this authorMarcia Ontell, Corresponding Author Marcia Ontell Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PennsylvaniaDepartment of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261Search for more papers by this author First published: 06 December 1998 https://doi.org/10.1002/(SICI)1097-0185(199705)248:1 3.0.CO;2-JCitations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Background Nuclear localizing β-galactosidase (nlsβ-gal) is used as a marker for studying myoblast cell lineage and for evaluating myoblast survival after myoblast transfer, a procedure with potential use for gene complementation for muscular dystrophy. Usefulness of this construct depends on the establishment of the extent to which nlsβ-gal or its mRNA may be translocated from the nucleus that encodes it to other noncoding myonuclei in hybrid myofibers and the ease with which the encoding and noncoding myonuclei can be distinguished. Previous in vitro studies (Ralston and Hall 1989. Science, 244:1066–1068) have suggested limited translocation of the fusion protein. We reexamined the extent to which nlsβ-gal is translocated in hybrid myofibers, both in vitro and in vivo, and evaluated the extent to which one can rely on histochemistry to distinguish encoding from noncoding nuclei in these myofibers. Methods Myotubes formed in cocultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nlsβ-gal under the control of the myosin light chain 3F promoter and 3′ enhancer (3FlacZ10 cells), and [3H]-thymidine-labeled parental MM14 cells (plated at ratios of 1:6 or 1:20, respectively) were reacted with X-gal. After autoradiography, the distance over which nlsβ-gal was translocated in hybrid myotubes was determined. In vivo translocation of nlsβ-gal was evaluated by injecting [3H]-thymidine-labeled 3FlacZ10 myoblasts into the regenerating extensor digitorum longus muscle of immunosuppressed normal and mdx (dystrophin deficient) mice. Sections stained with X-gal and subjected to autoradiography permitted determination of the extent of nlsβ-gal translocation in hybrid myofibers. Results In vitro: All nuclei in > 92% of hybrid myotubes showed evidence of nlsβ-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei ∼350 μm from a 3FlacZ10-derived myonucleus showed evidence of nlsβ-gal. In vivo: Similar translocation of nlsβ-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from host-derived myonuclei containing nlsβ-gal by the greater accumulation of reaction product in donor myonuclei after X-gal staining. However, 2 weeks after injection, host myonuclei often contained a significant amount of nlsβ-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei. Conclusions Translocation of nlsβ-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nlsβ-gal positive noncoding myonuclei in hybrid myofibers. One week after myoblast transfer, distinguishing between nlsβ-gal encoding and noncoding myonuclei in hybrid myofibers after X-gal stainbers. 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