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Fig. 1 | Molecular Brain

Fig. 1

From: Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

Fig. 1

Generation of control and PRKN-mutated patient TH-GFP iPSC lines via the CRISPR/Cas9 system. a Cas9 target sites in the exon 12 sequence of the human TH gene. Black, magenta, and cyan characters represent intron, exon, and UTR sequences in the human TH gene, respectively. The stop codon is shown in bold. b Scheme describing the insertion of the reporter cassette into the TH gene by homologous recombination. Green and magenta arrows indicate the primer pairs used for the detection of the TH-GFP and unedited alleles, respectively. c The differential interference contrast (DIC) and fluorescent images showed RFP-positive knock-in iPS colonies after 8–9 days of puromycin selection. “PRKN” represents PRKN-mutated patient. Scale bar, 500 µm. d PCR analysis of knock-in iPS clones with TH-GFP alleles to determine whether the clones were homozygous or heterozygous for the TH-GFP allele. The TH-GFP allele produced by cleavage at the Target 1 (T1) or Target 2 (T2) site was found as a 1.5 kb or 1.6 kb band, respectively. The unedited TH allele was found as a 1.0 kb band. e Sanger sequencing confirmed that the donor sequence was correctly inserted into exon 12 of the TH gene in four knock-in iPSC lines. The 1.2 kb PCR products by primers (black arrows) were sequenced, and the sequence of the junction of the 5′ homology arm and T2A-GFP at the cleavage site (boxed area) is indicated as the electropherograms. “PRKN” represents PRKN-mutated patient

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