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. 2017 Jun 8;7:3036. doi: 10.1038/s41598-017-02785-y

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© The Author(s) 2017

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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Application of the EGFP-KI reporter PFFs. (A) The purity of each EGFP gene site-specific knock-in cell clones was analysed via fluorescence microscopy and confirmed via FACS. (B) To confirm of the purity of each cell clone by western blotting (shown on the left); Genomic PCR analysis of the blastocysts (shown on the right); these 23 nuclear donor cells were derived from cell clone-EK4. The results show the high purity of cell clone-EK4. The red arrow indicates the knock-in bands and the blue arrow indicates the wild-type bands. NC: negative control. (C) The proportion of mixed nuclear donor cells was analysed via fluorescence microscopy and confirmed via FACS. (D) Genomic PCR analysis of the blastocysts (upper). Nuclear donor cells were derived from the mixed nuclear donor cells in fig. C . The red arrow indicates the knock-in bands. After 7 days of culture, the mixed blastocysts were observed by fluorescence microscopy (lower). M: D2000. (E) Generation of the EGFP reporter PK-15 cell lines was confirmed via fluorescence microscopy.