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. 2014 Sep;24(9):1526–1533. doi: 10.1101/gr.173427.114

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© 2014 Xie et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — Precise excision of piggyBac in mutation-corrected iPSC clones. (A) Primers P4/P8 and P6/P7 amplify the CAG promoter and puro∆TK, respectively. No amplification by both pairs indicates no piggyBac reintegration either at the targeting site or other chromosomal locations in clones 75-5 and 75-7. P1/P3 amplifies the junction between the piggyBac insert and the globin gene sequence to the 3′ genomic sequences. No amplification by these primers in these two clones indicates the removal of the piggyBac insert from HBB, while positive amplification (B) with P2/P9 further confirms PB removal. (C) Sequence analysis showing the junction between the ITR of the piggyBac and genomic sequences before transposon removal and the restoration of the normal intron 1 sequence after removal. Note the TTAA sequence of the piggyBac that is used for insertion and excision from the genome.