Generation of MYBPC3-KO hiPSCs. (IMAGE)
Caption
(A) The incidence data of MYBPC3 in hypertrophic cardiomyopathy were summarized, and the results were obtained from the ClinVar database. (B) The structure of the MYBPC3 gene and the location of gRNA for epiCRISPR/Cas9 editing. (C) Sequencing chromatograms verified a homozygous MYBPC3-KO hiPSC line, characterized by a deletion of 2 nucleotides in each allele. (D) Immunostaining of MYBPC3-KO colonies for the pluripotency markers OCT4 and SSEA4. Scale bar, 20 μm. (E) Molecule-based methods were used to induce cardiac differentiation. (F) Analysis for cTnT from WT and MYBPC3-KO differentiation protocols before purification at day 10. (G, H) Quantitative real-time PCR and western blotting analysis of MYBPC3 gene in both WT and MYBPC3-KO hiPSC-CMs at day 20. (I) Immunostaining for expression of MYL2 in WT and MYBPC3 KO CMs at day 40. Scale bar, 50 μm. The results were presented as mean ± standard deviation of three independent experiments. *P < 0.05; **P < 0.01; n.s. means no significance. MYBPC3, myosin binding protein C3; KO, knockout; WT, wild type; hiPSC, human induced-pluripotent stem cell; CM, cardiomyocyte; SSEA4, stage-specific embryonic antigen 4; OCT4, octamer-binding protein 4; cTnT, cardiac troponin T; MYL2, myosin light chain 2.
Credit
Jinhua Cao, Yafei Zhai, Ke Li, Jiajv Li, Xiaoxu Tian, Jianchao Zhang, Shuang Li, Mengduan Liu, Xiaowei Li, Jianzeng Dong, Xiaofang Wang
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