Xenbase Image ID: 153237

Figure 2: Shifting open reading frame upon inserting DNA into the MCS of the pmCherry-GFP construct leads to the loss of functional GFP moiety. (A) Diagram for cloning a genomic DNA fragment encompassing the region targeted by genome editing. Primers R and F are used to amplify a genomic fragment containing the targeted region. Two nested primers, NestF, with the recognition site by a restriction enzyme RE1 at the 5′-end, and NestR, with the recognition site by a restriction enzyme RE2 at the 5′-end, are designed to amplify a smaller fragment that is a continuous ORF in the wild type genome. The smaller fragment is purified and cleaved with RE1 and RE2 and dephosphorylated (to prevent self ligation of the fragment). The fragment is then cloned into pmCherry-GFP predigested with RE1 and RE2. The plasmid is then transformed into bacteria. If the PCR fragment is from wild type genomic DNA, a fusion protein of mCherry and GFP will be made in the bacteria. If genome editing leads to an in-frame mutation, a fusion protein of mCherry and GFP will also be made. If genome editing leads to an out-of-frame mutation or stop codon, the fusion protein will contain only mCherry but no functional GFP. (B). Diagram for cloning a genomic DNA fragment encompassing the region targeted by TALEN in the Xenopus tropicalis TRα gene. For the wild type genome, a fragment of 294 bp flanking the TALEN target site will be inserted into the pmCherry-GFP construct to produce a fusion protein with functional mCherry and GFP. (C). Inserting an out-of-frame but not an in-frame mutant fragment from the TRα gene disrupts GFP function. The PCR fragments amplified from the wild type DNA (Wt), two known in-frame deletion mutants (M11 and M15 with deletions of 3 and 15 bp, respectively) or an out-of-frame mutant (M16, with a 5 bp insertion) were cloned into pmCherry-GFP. The resulting plasmids were transformed into bacteria. The bacterial plates were visualized for mCherry and GFP. The merged mCherry and GFP images were shown in the last column. 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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