Fig. 5
From: Genome editing through large insertion leads to the skipping of targeted exon
Exon skipping can be abolished by minimizing the degree of alteration. a Exon 4 of hCDC14B was targeted by ZFN to introduce double strand break (DSB) and error-prone NHEJ for random insertion and deletion. In some of the targeted loci, small alterations in the exon (one to fifteen bases) were observed. ZFN cutting sites are written in blue font and base deletions or insertions are marked by red font color. Sequencing of the RT-PCR products confirmed identical base pair changes without exon skipping (sequence not shown). b The selection cassette flanked by loxP sites was removed by Cre-recombinase from HCT116 and RPE1 hCDC14B-KO cells. RT-PCR analysis confirmed the presence of stop codon (11 bp) followed by one loxP site (34 bp) within the targeted exon in both the cell lines. c Semi-quantitative RT-PCR indicated that the exon-skipped mRNA level of RPE1 cells is less than half of the wild-type mRNA. 40 ng of total RNA was used and the input RNA level was confirmed by GAPDH amplification as mentioned by Carbery et al. [30]. The experiment was performed three times with similar outcome. One representative experiment is shown. The numbers below the agarose gel summarizes the relative abundance of the mRNA from three different experiments