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Supplemental figure 4: Sequence analysis confirms that out-of-mutation rates determined from the two-color assay reflects the actual out-of-mutation rates within the TALEN targeted regions. The mRNAs encoding the left and right arms of a TALEN targeting the TRα-DBD (A), Sox3 (B), TRα-LBD (C) or Dot1L (D, TALEN1 and TALEN2, respectively) region shown in Fig. 2B, Fig. 3A and Supplemental Fig. 2A were injected into fertilized eggs and the animals were reared into tadpole stages. The genomic DNA was isolated from the tadpoles, PCR amplified, cloned into pmCherry-GFP (for TRα-DBD and Sox3) or pLacZα-GFP (for TRα-LBD and Dot1L TALEN1 and TALEN2, respectively) constructs and analyzed as indicated in Material and Methods. All colonies on a plate containing about 40 to 50 colonies for each target were picked up and grew in LB medium supplemented with ampicillin. The plasmid DNA was isolated and subjected to DNA sequencing. The TALEN targeted sequences are in blue and the areas are shaded. The mutations with deletions (-) and additions (+) were indicated. When there was more than one colony of a particular mutation, the number of colonies was shown in the parenthesis. Out-of-frame mutations were in bold letters. The out-of-frame mutation rates were calculated by dividing the numbers of out-of-frame mutants by the total clones containing the respective TALEN target sequences and summarized in Table 2. Image published in: Fu L et al. (2016) Copyright © 2016, The Author(s). 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 license Larger Image Printer Friendly View

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