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. 2019 Feb 13;9:1971. doi: 10.1038/s41598-018-37192-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Genotyping of transgene integration and orientation by PCR analysis. To map the orientation of the transgene integration events at the ROSA26 locus and to characterize indels at the 5′ and 3′ junctions of the insertion site, genomic DNA from F0 founders with observed en face tdTomato expression and non-transgenic wild-type ferrets were analyzed for transgene integration using a PCR assay. (A) Schematic of the PCR strategy and primers used for mapping the direction of integration events. (B) Representative gel images of PCR products (P1-P5) for the indicated transgenic (TG) founder ferrets and a non-transgenic wild-type (WT) animal (see Table 2 for primer descriptions). The predicted sizes of PCR products varied among distinct TG ferret founders due to different sized indels generated from error-prone NHEJ-mediated integration. The sizes of these indels were confirmed by Sanger sequencing of the PCR products (Fig. 4). M, lane denotes 1 kb plus marker.