Fig. 1

DIP2C knock-out by rAAV-mediated deletion of 48 bp in exon 9 in human cancer cells. a Coding sequence and predicted structural protein domains of DIP2C. Alternating exons are indicated and exon 9, targeted for deletion of 48 bp, shown in black. Triangles represent mutations found in cancer; black - breast cancer [4], grey - lung cancer [5], filled – missense mutation, open – frameshift mutation. Coding exons and domains from Ensembl ENST00000280886.11, UTRs not shown. b The targeting vector with regions homologous to the 5′ and 3′ ends of DIP2C exon 9 and surrounding intronic sequence includes a promoter trap selectable neo marker (IRES neo), LoxP sites (triangular) for selection cassette removal, and AAV inverted terminal repeat (ITR) sequences (dashed lines) mediating integration into the genome. Genomic DIP2C alleles are targeted through homologous recombination mediated by rAAV, as shown by dotted lines. Correct integrations are identified by PCR after enrichment by neo selection. Cre recombinase excises the selectable marker, leaving one LoxP sequence at the integration site. The targeted deletion leads to frameshift during translation. In addition STOP codons in the vector sequence ensure protein truncation (open grey triangles). Genomic locations of exons 8 and 9 are shown before and after targeting. Black lines – vector sequence, grey lines – genomic sequence. Primer sites are indicated by arrows. c-d Expression of DIP2C mRNA in DIP2C knockout (c) and overexpression (d) clones measured by RT-qPCR. Three biological replicates for parental RKO, two replicates for DIP2C ^−/− #1-1, and singleplex samples for the other clones were assayed in technical triplicates. Bars - relative quantity (RQ) minimum and maximum. e Western blot for DIP2C in DIP2C overexpression clones. Predicted MW 170.8 kDa. # - knockout clone, OE – overexpression clone, (*P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t-test)