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. 2021 Jan 28;11:2424. doi: 10.1038/s41598-021-81975-1

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Heterogeneous expression of multi-gene construct. (A) Schematic showing our multi-gene construct design in which expression of four genes (PuromycinR, mNeonGreen-NLS, mRuby3-NLS, and ZeocinR) is constitutively driven by independent CAG promoters. Genes are flanked by CTCF insulators (orange) and WPRE-bGHpolyA (pA) sequences. The multi-gene transcripts are then flanked by 5′ and 3′ homology arms (HA) directing insertion into the AAVS1 loci. (B) Fluorescent imaging representative of the four stable and distinct phenotypic populations observed after undergoing multiple rounds of puromycin selection at 14 days post-transfection. DN Double negative, G mNeonGreen-NLS only, R mRuby3-NLS only, DP Double Positive.