Xenbase Image ID: 128656

Fig. 3. Disruption of pax6 in the TALEN mRNA-injected embryos.(A) Phenotypes of pax6 TALEN-injected embryos: TALEN-R, embryos injected with 1100 pg right pax6 TALEN mRNA; TALEN-R/L, embryos injected with 550 pg of each right and left pax6 TALEN mRNA. Injected embryos were reared to the hatching stage. Upper image: low magnification of each experimental group. Lower image: high magnification of representative phenotypes from TALEN-R- and TALEN-R/L-injected embryos noted by squares in the upper image. (B) Detection of pax6 mutations by genomic PCR and digestion with a restriction enzyme. One embryo was collected from each of the three experimental groups (asterisks in panel A): Uninjected, TALEN-R, and TALEN-R/L. Upper image: a schematic drawing of pax6 genomic PCR product. Genomic DNA of each embryo was prepared and then was subjected to PCR with a specific primer set for amplification containing the pax6 TALEN target site. Hpy188I site is located on spacer sequence of the TALEN site. Left image: a gel electrophoresis image of pax6 genomic PCR products. Right image: Hpy188I-digestion of PCR products for detecting pax6 mutations. PCR products were purified and then digested by Hpy188I. Hpy188I fragments are divided into 101 and 54 bp. M means 100 bp ladder marker. Uninjected indicates uninjected control embryo. (C) Sequences and frequencies of pax6 mutation by TALENs. PCR product from the TALEN-R/L-injected embryo (asterisk in panel A) was subcloned and was subjected to DNA sequencing analysis. Mutant sequences were aligned to those of wild type. Sequences of pax6a and pax6b wild-type alleles are shown at the top. Yellow boxes indicate pax6 TALEN target site. Gaps generated by deletion are shown as blue dashes. Types and frequencies of each mutation (deletion and insertion) are shown at the right of the panel. Paralogous substitutions between pax6a and pax6b are highlighted in green. Image published in: Suzuki KT et al. (2013) © 2013. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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