Xenbase Image ID: 153238

Figure 3: Quantifying out-of-frame mutation rate in F0 generation tadpoles produced with a TALEN targeting TRα gene in Xenopus tropicalis by using pmCherry-GFP construct.The mRNAs encoding the left and right arms of a TALEN targeting TRα as shown in Fig. 2B were injected into fertilized egg and the animals were reared into tadpole stage (see16 for details). A number of the resulting tadpoles (M) were euthanized together and the total genomic DNA was isolated. Genomic DNA was also isolated from wild type tadpoles (Wt). The TRα region from the Wt and M tadpoles was cloned into pmCherry-GFP and analyzed as shown in Fig. 2. Note that the yellow colonies in the merged panels represented the colonies with wild type TRα DNA inserted into pmCherry-GFP or colonies with in-frame mutations in the TRα DNA inserted into pmCherry-GFP. The red colonies in the merged image represented colonies with out-of-frame mutations in the TRα DNA inserted into pmCherry-GFP. *The single red colony from wild type genomic DNA plate. Sequencing of the colony revealed a point mutation leading to an in-frame stop codon in the TRα fragment, likely due to polymorphism at the site in the Xenopus genome or mutation introduced by PCR amplifications. Based on the number of red and yellow colonies in the merged panel, the out-of-frame mutation rate in the TRα gene due to TALEN can be determined (see Table 2 and text for details). 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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