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. 2020 Aug 22;12(9):2378. doi: 10.3390/cancers12092378

Figure 2.

Figure 2

Genotyping analysis of CRISPR/Cas9 edited GC cells. (A) Analysis of gDNA (Panel 1) and cDNA (Panel 2) from wild-type (WT), Mock and edited GC cell lines. In Panel 1, MKN45/GP202_Mock cells present a transcript of the same size of WT (422 bp), while the edited cell lines present a transcript with smaller sizes (MKN45_O26 and GP202_O26–186 bp; MKN45_O15–293 bp; GP202_O25–232 bp). In Panel 2, MKN45/GP202_WT and Mock cells present a transcript with a size of 378 bp, while all edited cell lines present a smaller transcript (249 bp) corresponding to the occurrence of exon-v6 skipping. All PCR products were run in the same 2% agarose gel (Figure S1). (B) Representative examples of sequencing analysis: Panel 1—gDNA of Mock cells; Panel 2—gDNA deletion of 235 bp of O26 clones. (The present sequencing analysis was performed with gDNA extracted from MKN45_O26, but GP202_O26 presents the same deletion); Panel 3—gDNA deletion of 131 bp of O15 clone (* C nucleotide is a single nucleotide insertion); Panel 4—gDNA deletion of 190 bp of O25 clone; Panel 5—cDNA of Mock cells; Panel 6—cDNA of all edited clones, which represents the skipping of exon-v6. (C) Specifications of each edited cell line by CRISPR/Cas9 with the respective edition genomic coordinates (GRCh38/hg38) and the combination of sgRNAs transfected to create each edited cell line. WT–wild-type; M–Mock; gDNA–genomic DNA; cDNA–complementary DNA; Chr—Chromosome; Del—Deletion; Ins—Insertion.