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. 2017 Oct 6;7:12739. doi: 10.1038/s41598-017-10418-7

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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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Representative PCR and FISH results of CHO cells containing the modified hChr.21. (a) Representative results of transgene insertion analysed by PCR (red for positive clones). The negative control (N.C.) was CHO (hChr.21), which contained intact hChr.21. The positive control (P.C) was a CHO clone containing 21HAC2, targeted with I-EGFP-I-Bsd in DT40 cells, as previously described¹⁰. (b) Representative results of telomere-associated truncation analysed by PCR (red for positive clones with primer pair #3). N.C. was CHO (hChr.21) and P.C. was a CHO clone containing 21HAC2. The red boxed lane shows the clone showing an ideal result (HisD #44). (c) Representative FISH images of transgene insertion. Panels on upper lane show an intact hChr.21 in CHO cell. Panels on under lane show a hChr.21 with the inserted transgene in CHO Bsd #36. The rhodamine (red) signal indicates the repetitive sequence of human Cot-1 for staining of whole human chromosome. The FITC signal (green), which was observed as a yellow dot, indicates the presence of the inserted I-EGFP-I-Bsd-Fcy; Fur vector on the near centromeric region. The chromosome size was similar between the intact hChr.21 and the transgene-inserted hChr.21. (d) Representative FISH images of telomere-associated truncation. The upper panels show an intact hChr.21 in CHO cell. The under panels show a truncated hChr.21 in CHO HisD #44. The rhodamine (red) signal indicates the human Cot-1 for staining of whole human chromosome. The FITC signal (green) indicates the presence of pBS-TEL/Dq-HisD vector in the terminal end of the truncated hChr.21. The chromosome size of the truncated hChr.21 in CHO HisD #44 was much smaller than the intact hChr.21. (e) Representative images of quinacrine hoechst staining. Left pair: a smallest CHO chromosome and a morphologically intact hChr.21 in CHO (hChr.21), right pair: a smallest CHO chromosome and a truncated hChr.21 in CHO HisD #44. The deletion of q11-terminal end was observed on the truncated hChr.21. Full-length gels of the PCR analysis are presented in Supplementary Figure 1.