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. 2020 May 1;11:2141. doi: 10.1038/s41467-020-16030-0

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© The Author(s) 2020

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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Fig. 2 — a Schematic representation of PA-Cre components indicating codon modification site, 2A cleavage site and Cre antibody recognition site. b Representative western blotting images to compare CMV and CAG promoters for testing Magnets and Magnets codon-optimized PA-Cre (Magnets-opti). All protein expressions were accomplished in the dark condition (single independent experiments). c Comparisons of the original Magnets- and Magnets-opti-based PA-Cre using CMV or CAG promoter with 2A or IRES sequences (Magnets-IRES, Magnets-opti-IRES) using dual Luc assay. Luc assays were conducted in HEK 293T with double-floxed inverted Fluc. The upper diagram shows an illumination protocol used for the assays (blue LED, 447.5 nm, 8.28 W/m², repeated 20 s light and 60 s dark for 12 h) (n.s. not significant, ***P ≦ 0.0005; ****P < 0.0001; dark v.s. BL using Two-tailed t test, Biologically independent samples; Mock, CMV-Magnets: n = 13, CMV-Magnets-IRES n = 11, CMV-Magnets-opti n = 9, CMV-Magnets-opti-IRES, CAG-Magnets, CAG-Magnets-IRES: n = 6, CAG-Magnets-opti n = 9, CAG-Magnets-opti-IRES2 n = 8, mean ± s.d.). Red values show fold induction of Luc with BL compared with dark. d Comparison of CAG-driven Magnets-opti-based PA-Cre (PA-Cre 3.0) with the original version of PA-Cre (Magnets-based) using fluorescence imaging in mouse livers in vivo. Fluorescence liver imaging of tdTomato expression was conducted in Rosa26^Ai14/WT (Ai14: Floxed-tdTomato heterozygote) live mice transiently injected with CAG-Magnets or CAG-Magnets-opti plasmids, respectively in the dark, ambient light and under blue-light illumination (470 ± 20 nm, 200 W/m², 16 h continuous) (n = 2–6 mice/group, Scale bar: 1 mm). White arrowheads show tdTomato-positive cells in the livers in the dark and ambient light conditions. Source data are provided as a Source Data File.