We sought here to induce the excision of a large intragenic segment within the intact dystrophin gene locus, with the ultimate goal to elucidate dystrophin protein function and stability in striated muscles in vivo. To this end, we implemented an inducible-gene excision methodology using a floxed allele approach, demarcated by dystrophin exons 2–79, in complementation with a cardiac and skeletal muscle directed gene deletion system for spatial–temporal control of dystrophin gene excision in vivo. Main findings of this study include evidence of significant intact dystrophin gene excision, ranging from ~ 25% in heart muscle to ~ 30–35% in skeletal muscles in vivo. Results show that despite evidence of significant dystrophin gene excision, no significant decrease in dystrophin protein content was evident by Western blot analysis, at three months post excision in skeletal muscles or by 6 months post gene excision in heart muscle. Challenges of in vivo dystrophin gene excision revealed acute deleterious effects of tamoxifen on striated muscles, including a transient down regulation in dystrophin gene transcription in the absence of dystrophin gene excision. In addition, technical limitations of incomplete dystrophin gene excision became apparent that, in turn, tempered interpretation. Collectively, these findings are in keeping with earlier studies suggesting the dystrophin protein to be long-lived in striated muscles in vivo; however, more rigorous quantitative analysis of dystrophin stability in vivo will require future works in which more complete gene excision can be demonstrated, and without significant off-target effects of the gene deletion experimental platform per se.
Bibliographical noteFunding Information:
The authors acknowledge financial support from NIH (R01HL138490) and MDA of JMM and from the Paul D. Wellstone Muscular Dystrophy Specialized Research Center Grant (1U54NS053672) to KPC. KPC is an investigator of the Howard Hughes Medical Institute. We also thank Jason Mitchell from the University of Minnesota Imaging Center for technical support on Vevo 2100 System.
© 2020, The Author(s).