Wga alexa488 conjugate

Snap-frozen heart tissue was sectioned by cryotome and counterstained with DAPI and WGA-Alexa488 conjugate (Thermo Fisher Scientific). MCherry signal, indicating successful transduction, was visualized with the Thunder imaging system (Leica). Genomic DNA from heart tissue was isolated by Proteinase K (Carl-Roth) digestion and phenol-chloroform extraction. Deletion of the fragment between the MYBPC3_Ex6 and MYBPC3_Ex23 gRNA target sites was screened by PCR using Q5 polymerase (New England Biolabs) and flanking primers ∼0.6 kb distal to the gRNA target sites (Table 1). Correctness of amplified bands was confirmed by Sanger sequencing. Efficiency of editing was determined by qPCR using Viia7 Real-time PCR System (Applied Biosystems). Briefly, MYBPC3 wild-type exon 6 and the deletion variant (g41575_16906 del) were amplified by PCR using the primers MYBPC3_Ex6_F (5′-GCC​CCA​AGT​CTC​CTC​TAA​CA-3′) and MYBPC3_Ex6_R (5′-TCA​CCT​ATT​CAC​CTG​TGC​CC-3′) for exon 6 and MYBPC3_Ex6_F and MYBPC3_Del_R (5′-CAA​TGG​GCA​TGA​AGG​GCT​G-3′) for the deletion variant. Amplicons were quantified photospectometrically, standard curves were generated by serial dilution, and initial quantities of both amplicons in the tissue sample were determined by qPCR using the primers above. The frequency of the deletion variant was obtained by calculating the ratio of wild-type exon 6 to the deletion variant.