Fig. 2
From: Genome editing through large insertion leads to the skipping of targeted exon
Southern blot hybridization to confirm biallelic targeting of hCDC14A and hCDC14B loci in RPE1 cells. a Map for hCDC14A and hCDC14B genomic locus targeted by zinc finger nuclease (ZFN). The probes for Southern blot hybridization were designed in the right homologous arms (shown as green bar). In case of hCDC14A, digestion by Hind III would result in a 2.9 kb fragment for knockout cells instead of wild-type 1.2 kb band. For knocking out hCDC14B, neomycin or puromycin inserted donor templates were used. Puromycin construct (with an extra Hind III site) was used when hCDC14B knocking out was carried out on top of hCDC14A −/− cells (NeoR) (double knockout). On the other hand, neomycin construct was used during generation of single hCDC14B knockout. b Southern blot hybridization to confirm hCDC14A knockout (AKO) with a hCDC14A specific probe (a). The expected hCDC14A band sizes were observed for wild type (Wt, 1.2 kb), hCDC14A single (AKO) and hCDC14A hCDC14B double knockouts (DKO) (2.9 kb). c Southern blot hybridization to confirm hCDC14B knockout (BKO) with a hCDC14B specific probe (a). The DKO (see a, PuroR) and Wt cells have shown anticipated band sizes of 3.2 kb and 4.0 kb, respectively. For single hCDC14B knockouts (BKO), two extra bands above and below the expected band size of 5.7 kb (asterisks) was persistently observed in different clones. Absence of wild type bands (4.0 kb) in these knockouts confirmed the successful targeting of both alleles