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. 2023 May 25;19(7):887–899. doi: 10.1038/s41589-023-01344-z

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© The Author(s) 2023

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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Extended Data Fig. 1 — (a) Strategy to validate the insertion specificity of LP cassette of each monoclonal sample via PCR genotyping. Primers were designed to amplify the adjunction (border) regions between the constructs and the genome at both the (5’ and 3’) ends of the inserts. The adjunction regions with the vector backbones were also tested for any possible random integration. Identical primers are indicated in the same color. (b) PCR genotyping of 293 and MB231 Landing Pad (LP) cell lines using primer sets depicted in (a) for 5’/3’ junction assay (JA) and random integration assay (RI). Parental cells (WT) and Donor plasmid were included as controls. (c) EeGFP copy number determination via qPCR of the selected 293 and MB231 LP cell lines. Each sample copy number was normalized to the reference gene RPPH1 (n = 3). (d) Identical constitutive GFP expression of representative LP cell lines of 293 and MB231 over time (n = 3). (e) Mean fluorescence intensity (MFI) comparison between parental Landing Pad cells, versus uninduced mNF-GFP and mNF-BACH1 integrated cells. n = 3, One-way ANOVA, P < 0.0001. (f) Strategy to validate the insertion specificity of mNF circuits for each monoclonal sample via PCR genotyping. Primers were designed to amplify the adjunction regions between the constructs and the genome at both the (5’ and 3’) ends of the inserts. The adjunction regions with the vector backbones were also tested for any possible random integration. Identical primers are indicated in the same color. (g) PCR genotyping of the mNF-BACH1- (purple) and mNF-GFP- (green) integrated monoclonal populations and polyclonal populations (marked as clone 0) of 293-3 and MB231-1 using primer sets depicted in (f) for 5’/3’ junction assay (JA) and random integration assay (RI). Parental Landing Pad cells (LP) and mNF circuit donor plasmids were included as controls. (h) eGFP copy number determination via qPCR of the selected 293 (left) and MB231 (right) mNF-BACH1- (purple) and mNF-GFP- (green) integrated monoclonal populations relative to the corresponding parental Landing Pad population (LP, grey). Each sample was normalized to corresponding LP sample in each cell type. One-way ANOVA, n = 3.

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