Xenbase Image ID: 153240

Figure 5: Substitution of mCherry coding region with that of LacZα allows detection of out-of-frame mutations with a combination of X-gal staining and a simple fluorescent microscope (without a need to resolve red vs. green fluorescence). A PCR fragment flanking the TALEN targeting site in TRα gene in Xenopus tropicalis was amplified with the NestF(BamHI) and NEstR(EcoRI) from genomic DNA isolated from a wild type tadpole (Wt), a tadpole with a known in-frame deletion (M15 with a deletion of 15 bp) or an out-of-frame mutant (M16, with a 5 bp insertion) shown in Fig. 2C, or mutant F0 TRα TALEN tadpoles (Fig. 3). The amplified fragment was cloned into the BamHI and EcoRI double-digested pLacZα-GFP (same as pmCherry-GFP except the mCherry sequence has been replaced with LacZα sequence, illustrated on top). The resulting plasmids were transformed into bacteria. The bacterial plates were stained with X-gal. Pictures were taken under the bright field (Bright Field) to visualize X-gal staining (blue) or under a fluorescent microscope for GFP. Note that colonies from plasmid containing Wt DNA or DNA with the in-frame deletion of 15 bp were greenish blue under the bright field, likely reflecting the blue color of the X-gal staining plus weak green color due to the GFP under visible light. On the other hand, a 5 bp insertion caused frame shift and disrupted GFP function, leading to pure blue colonies in the bright field or merged images. Colonies from the mutant tadpole (M) were a mixture of blue (lack of GFP) or greenish blue colonies in the bright field. 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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