Xenbase Image ID: 152309

Figure 4. Genotyping of founders with different phenotypes with RFLP analyses. (A) A schematic of restriction enzyme-RFLP (RE-RFLP) and RNA-guided engineered nuclease-RFLP (RGEN-RFLP) analysis. (B) Estimation of somatic mutation rates of the target alleles in each phenotype. PCR products of tyr and ltk were cleaved by Cas9 protein with sgRNAs in vitro. PCR products of slc45a2 were cleaved by BfaI. Cleaved fragments were analyzed by electrophoresis (MultiNA system; Shimadzu). Upper and lower images show Cas9 protein or BfaI (+) and (−) gel images converted from electropherograms. PCR products of target regions are shown by black arrowheads. Colored arrowheads indicate cleaved products by Cas9 or BfaI. Some extra bands were observed and thought to be large deletions and insertions. The mutation rates were calculated from the molarity of the uncleaved bands (black arrowheads and other extra bands) and the larger cleaved fragments (blue arrowheads). Wt, wild type; W, weak phenotype; M, moderate phenotype; S, severe phenotype; PS, severe phenotype by Cas9 protein injection; H, half phenotype; F, full phenotype. Full scan images of RFLP analyses are shown in Figs S5–S8 in Supporting Information. Image published in: Shigeta M et al. (2016) Copyright © 2016. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View

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