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. 2022 Jul 8;50(13):7783–7799. doi: 10.1093/nar/gkac587

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© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Safe-harbour large cargo integration of NPHS2 into patient-derived R138Q mutant podocin podocytes. (A) Safe harbour ACTB-NPHS2-Myc-Flag strategy. ACTB C-terminal exon is replaced with a P2A mCherry T2A Puro tagged synthetic exon (5′ integration marker), followed by CMV NPHS2-Myc-Flag and CMV Hygro (3′ integration marker). polH VSV-G and CMV mTagBFP are included in vector design to pseudotype and monitor viral transduction, respectively. (B–E) PM ciPods transduced with BV MultiMate HITI-2c NPHS2 in presence/absence of 24 h BacMam enhancer treatment analysed at 24 or 72 h post transduction. (B) transduction efficiencies (mTagBFP+ %); (C) mTagBFP mean fluorescence intensity levels, (D) absolute gene editing efficiencies (mCherry+ %). In (B–E) histograms represents means of flow-cytometry data, error bars are standard deviations of n = 3 independent replicates. (E) representative flow-cytometry histogram of mTagBFP intensity relative to (C). (F) Widefield microscopy images of PM ciPods at 24 h post-transduction in presence/absence of BacMam enhancer (top panel) or 36 days post-transduction following Puromycin/Hygromycin selection (bottom panel). (G) Immunofluorescence of unselected PM ciPods transduce with BV MultiMate HITI-2c NPHS2. Successfully edited (mCherry+) cells, display correct expression of NPHS2 through either α-Flag or α-NPHS2 antibodies. DAPI is used to counterstain nuclei. Scalebar = 20 μm. (H) Comparison of engineered PM ciPods with WT CiPods overexpressing NPHS2 R138Q under proliferative (33°C, top panel) or non-proliferative (10 days at 37°C, bottom panel) culturing conditions. α-Calnexin labels the endoplasmic reticulum, α-Myc is used to stain NPHS2. Scalebar = 20 μm.