Xenbase Image ID: 155269

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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