Limitations of nlsβ-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer
✍ Scribed by Yang, Jiwei ;Ontell, Martin P. ;Kelly, Robert ;Watkins, Simon C. ;Ontell, Marcia
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 200 KB
- Volume
- 248
- Category
- Article
- ISSN
- 0003-276X
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✦ Synopsis
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.