CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and VersatileAssessment of Myocardin

Objective –—Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, Myocardin (MYOCD). Accordingly, the mouse Myocd locus was targeted with two independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results –—3-component CRISPR was used to engineer a carboxyl-terminal 3xFLAG or 3xHA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a protein product of ~150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in heart. Both alleles of Myocd are active in aorta since a two-fold increase in protein was seen in mice homozygous versus heterozygous for FLAGtagged Myocd. ChIP-qPCR studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged VSMC exhibited expected nuclear localization. Conclusion –—This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research. To whom correspondence should be addressed: Joseph M. Miano, PhD, Aab Cardiovascular Research Institute, University of Rochester School of Medicine & Dentistry, 601 Elmwood Avenue, Rochester, NY 14624, Phone: 585-276-7703, Fax: 585-276-9830, j.m.miano@rochester.edu. Disclosures None. HHS Public Access Author manuscript Arterioscler Thromb Vasc Biol. Author manuscript; available in PMC 2019 September 01. Published in final edited form as: Arterioscler Thromb Vasc Biol. 2018 September ; 38(9): 2184–2190. doi:10.1161/ATVBAHA. 118.311171. A uhor M anscript