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. 2018 Jul 10;8:10385. doi: 10.1038/s41598-018-28502-x

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© The Author(s) 2018

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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Profiles of transgene expression from the bRosa26 locus. (A) TAT-Cre-mediated recombination induces EGFP expression. (B) PCR analysis of transgenic cloned cattle. Primers P5 and P6 amplified a 1.6 kb product, confirming the production of positive bRosa26-EGFP knock-in cloned cattle. Primers P7 and P8 amplified a 6.1 kb product and confirmed the production of positive bRosa26-EGFP knock-in cloned cattle. M, 1 kb DNA ladder; Lanes 2–5, genomic DNA from bRosa26-EGFP knock-in cloned cattle; P, donor vector; H2O and WT, negative controls. (C) Southern blot identification of transgenic cloned cattle. Upon digestion with DraIII or ScaI, a band of 9.2 kb or 5.8 kb was detected in transgenic cloned cattle, respectively. C1-C4, genomic DNA from bRosa26-EGFP knock-in cloned cattle; WT, wild-type cattle. (D) EGFP expression in various tissues from bRosa26-EGFP cattle relative to GAPDH expression, as determined by Q-PCR. Error bars represent the mean ± SD. (E) EGFP expression in various tissues from the bRosa26-EGFP cattle, as determined by RT-PCR. For RT-PCR, the designed primers annealed to the EGFP and amplified a correctly spliced product of 347 bp. GAPDH served as a control (488 bp). (F) EGFP expression in various tissues from the bRosa26-EGFP cattle, as determined by Western blot, GAPDH served as a control.