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. 2021 Jan 12;11:736. doi: 10.1038/s41598-020-80436-5

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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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Efficient puromycin selection of mosquito cells transfected with hSpCas9-pAc. (a) Aag2 cells were pre-treated with transfection reagent alone (puro), transfected with a control PUb-eCFP (enhanced cyan fluorescent protein) plasmid lacking a puromycin (puro) resistance cassette (puro + eCFP), or transfected with PUb-hSpCas9 linked to a puro resistance cassette by the T2A skipping site (hSpCas9-pAc). Day 2 post-transfection, cells were treated with 2.5 µg/mL puro. p < 0.0001 (overall ANOVA comparing different transfections); individual groups were compared using the Dunnett's post hoc test compared to the puro control; **p < 0.001, ***p ≤ 0.0001. (b) As in (a) for U4.4 cells treated with 10 µg/mL puro. *p < 0.05, ***p ≤ 0.0001. (c) Immunoblot of Aag2 cells treated with transfection reagent alone (ctrl) or transfected with hSpCas9 or hSpCas9-pAc (driven by PUb promoter). Expected size of hSpCas9 is ~ 160 kilodaltons (kDa); expected size of unprocessed hSpCas9-pAc is ~ 185 kDa. Full-length blots in Supplementary Fig. 4. (d) As in (c) for U4.4 cells.