CRISPR-Cas9-mediated epitope tagging provides accurate and versatile assessment of myocardin-brief report

Qing Lyu, Vidhi Dhagia, Yu Han, Bing Guo, Mary E. Wines-Samuelson, Christine K. Christie, Qiangzong Yin, Orazio J. Slivano, Paul Herring, Xiaochun Long, Sachin A. Gupte, Joseph M. Miano

Research output: Contribution to journalArticle

Abstract

Objective-Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results-3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD 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 the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction 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 vascular smooth muscle cells exhibited expected nuclear localization. Conclusions-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.

Original languageEnglish (US)
Pages (from-to)2184-2190
Number of pages7
JournalArteriosclerosis, thrombosis, and vascular biology
Volume38
Issue number9
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

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Clustered Regularly Interspaced Short Palindromic Repeats
Epitopes
Proteins
Chromatin Immunoprecipitation
Smooth Muscle
Antibodies
myocardin
Vascular Smooth Muscle
Smooth Muscle Myocytes
Aorta
Publications
Biomedical Research
Myocardium
Embryonic Structures
Western Blotting
Alleles
Genome
Muscles
Polymerase Chain Reaction

Keywords

  • allele
  • epitope
  • mice
  • muscle, smooth
  • myocardin

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

CRISPR-Cas9-mediated epitope tagging provides accurate and versatile assessment of myocardin-brief report. / Lyu, Qing; Dhagia, Vidhi; Han, Yu; Guo, Bing; Wines-Samuelson, Mary E.; Christie, Christine K.; Yin, Qiangzong; Slivano, Orazio J.; Herring, Paul; Long, Xiaochun; Gupte, Sachin A.; Miano, Joseph M.

In: Arteriosclerosis, thrombosis, and vascular biology, Vol. 38, No. 9, 01.01.2018, p. 2184-2190.

Research output: Contribution to journalArticle

Lyu, Q, Dhagia, V, Han, Y, Guo, B, Wines-Samuelson, ME, Christie, CK, Yin, Q, Slivano, OJ, Herring, P, Long, X, Gupte, SA & Miano, JM 2018, 'CRISPR-Cas9-mediated epitope tagging provides accurate and versatile assessment of myocardin-brief report', Arteriosclerosis, thrombosis, and vascular biology, vol. 38, no. 9, pp. 2184-2190. https://doi.org/10.1161/ATVBAHA.118.311171
Lyu, Qing ; Dhagia, Vidhi ; Han, Yu ; Guo, Bing ; Wines-Samuelson, Mary E. ; Christie, Christine K. ; Yin, Qiangzong ; Slivano, Orazio J. ; Herring, Paul ; Long, Xiaochun ; Gupte, Sachin A. ; Miano, Joseph M. / CRISPR-Cas9-mediated epitope tagging provides accurate and versatile assessment of myocardin-brief report. In: Arteriosclerosis, thrombosis, and vascular biology. 2018 ; Vol. 38, No. 9. pp. 2184-2190.
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