Skip to main content
. Author manuscript; available in PMC: 2018 Jun 9.
Published in final edited form as: Circ Res. 2017 Mar 29;120(12):1874–1888. doi: 10.1161/CIRCRESAHA.116.310283

Figure 1. CASAAV-based somatic mutagenesis efficiently depleted Jph2 in CMs during CM maturation in vivo.

Figure 1

(A) Workflow for CASAAV. (B) Depletion of tdTomato by AAV9 delivery of specific gRNA plus Cre to activate Cas9-P2A-GFP. Dot plot shows that most GFP+ cells were tdTomato negative. As a control, AAV9 delivery of Cre without gRNA activated tdTomato in almost all GFP+ cells (see also Online Fig. II). (C) Design of Jph2 gRNAs that target the first coding exon of the Jph2 gene. (D) Immunostaining of JPH2 in isolated CMs demonstrated efficient depletion of JPH2 in Cas9GFP+ CMs (arrow). Scale bar, 10 μm. (E) Quantification of the fraction of JPH2-depleted CMs among GFP− and GFP+ CMs. >50 CMs were counted per heart. n = 5 hearts. Student’s t-test: *p<0.05; **p<0.01; ***p<0.001. Non-significant p value is shown in parentheses. Plots show mean ± SD. (F–G) Depletion of JPH2 in transduced (GFP+) CMs. Gating strategy for FACS purification of isolated GFP+ CMs is shown in F. Panel G shows Western blot analysis of FACS-sorted GFP+ cells.