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. 2017 May 19;7:2193. doi: 10.1038/s41598-017-02460-2

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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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Tri-cistronic 2A constructs: positional effects on gene expression. (A) Tri-cistronic constructs in a P2A-T2A backbone expressing three FPs GFP, Td and i670 in different orders. (B and C) MEF-T cells were transduced with retroviruses (pMXs) encoding the different 2A constructs for 3 days. Live fluorescent images of these cells were taken at 20X (B) and then cells were collected for (C) western blotting analysis for GFP expression, or (D) flow cytometry analysis for dMFI of GFP (green bar), Td (red bar), and i670 (purple bar). (C) GFP was quantified and normalized to the β-actin loading control. (D) dMFI was normalized to that of GFP-Td-i670 for GFP, Td-i670-GFP for Td, and i670-Td-GFP for i670. (E) The same as (D) except performed in TTF. Mean ± SEM of multiple experiments were shown. Results from one-way ANOVA and bonferroni correction were summarized in a table. Scale bar = 200 µm.