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

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  • Author(s): Yang J;Yang J; Ontell MP; Kelly R; Watkins SC; Ontell M
  • Source:
    The Anatomical record [Anat Rec] 1997 May; Vol. 248 (1), pp. 40-50.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: A.R. Liss [etc.] Country of Publication: United States NLM ID: 0370540 Publication Model: Print Cited Medium: Print ISSN: 0003-276X (Print) Linking ISSN: 0003276X NLM ISO Abbreviation: Anat Rec Subsets: MEDLINE
    • Publication Information:
      Original Publication: [New York, etc.] A.R. Liss [etc.]
    • Subject Terms:
      Cell Transplantation*; Muscle, Skeletal/*cytology ; Muscle, Skeletal/*enzymology ; beta-Galactosidase/*metabolism; Animals ; Biological Transport, Active ; Biomarkers ; Cell Line ; Cell Nucleus/enzymology ; Female ; Humans ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/physiology ; Muscular Dystrophy, Animal/enzymology ; Muscular Dystrophy, Animal/genetics ; Muscular Dystrophy, Animal/therapy ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Rats ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Regeneration ; Transfection ; beta-Galactosidase/genetics
    • Abstract:
      Background: Nuclear localizing beta-galactosidase (nls beta-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 nls beta-gal or its mRNA may be translocated from the nucleus that encodes it to other non-coding myonuclei in hybrid myofibers and the ease with which the encoding and non-coding 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 re-examined the extent to which nls beta-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 non-coding nuclei in these myofibers.
      Methods: Myotubes formed in co-cultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nls beta-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 nls beta-gal was translocated in hybrid myotubes was determined. In vivo translocation of nls beta-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 nls beta-gal translocation in hybrid myofibers.
      Results: In vitro: All nuclei in > 92% of hybrid myotubes showed evidence of nls beta-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei approximately 350 microns from a 3FlacZ10-derived myonucleus showed evidence of nls beta-gal. In vivo: Similar translocation of nls beta-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from host-derived myonuclei containing nls beta-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 nls beta-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei.
      Conclusions: Translocation of nls beta-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nls beta-gal positive non-coding myonuclei in hybrid myofibers. One week after myoblast transfer, distinguishing between nls beta-gal encoding and non-coding myonuclei in hybrid myofibers after X-gal staining of sectioned muscle is feasible; however, by 2 weeks, nls beta-gal increases in host myonuclei, making identification of donor-derived myonuclei problematic. Translocation of nls beta-gal to non-coding myonuclei in hybrid myofibers must be considered when nls beta-gal is used for studies of myogenic lineage or the efficacy of myoblast transfer therapy, particularly if long-term survival of hybrid myotubes is required.
    • Grant Information:
      AR36294 United States AR NIAMS NIH HHS
    • Accession Number:
      0 (Biomarkers)
      0 (RNA, Messenger)
      0 (Recombinant Fusion Proteins)
      EC 3.2.1.23 (beta-Galactosidase)
    • Publication Date:
      Date Created: 19970501 Date Completed: 19970718 Latest Revision: 20190115
    • Publication Date:
      20240104
    • Accession Number:
      10.1002/(SICI)1097-0185(199705)248:1<40::AID-AR5>3.0.CO;2-J
    • Accession Number:
      9143666