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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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Quad-cistronic 2A constructs: example of application in cellular reprogramming. (A) Quad-cistronic constructs using different orders of 2As (PTE2A, TPE2A, and 3P2A) for the expression of Mef2c (M), Gata4 (G), Tbx5 (T) and Tdtomato (Td). A total of 12 constructs were built with Td rotating from position 1 to 4. (B) Flow cytometry quantification of the percentage of Td+ cells (red triangle) and dMFI of Td (purple diamond) in various cell types transduced (except transfection in 293T) with retroviruses (pMXs) encoding the different 2A constructs. dMFI of Td was normalized to that in the PTE2A-TdMGT construct. Cells were collected on day 3 post-transduction or 48 hr post-transfection (293T). (C) Western blots for M, G, T expression in PTE2A and TPE2A constructs. Right panel: quantification after normalization to the β-actin loading control. (D) Positional effects on protein expression in quad-cistronic constructs calculated based on (C). (E–G) Td expression and reprogramming with different 2A constructs in MEF-T on day 3 post-transduction. (E) Representative live fluorescent images of Td and the reprogramming reporter αMHC-GFP. All taken at 20X. Scale bar = 200 µm. (F) Flow cytometry quantification of Td and GFP single- and double-positive cells. (G) GFP dMFI in αMHC-GFP+ cells (blue bar) and the product of the percentage of αMHC-GFP+ cells and dMFI-GFP (green bar). dMFI of GFP was normalized to that of the TPE2A-MGTTd construct. Mean ± SD of triplicate experiments were shown in (B,D,E).