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. 2023 Jun 8;14:3369. doi: 10.1038/s41467-023-39137-6

Fig. 3. TJ-PE mediates GFP reporter and functional gene insertion.

Fig. 3

a A diagram of the TLR-MCV1 reporter line. Inserting an 89-bp sequence to replace the 39-bp non-functional sequence results in GFP expression. Indels result in mCherry expression. Del: deletion. b PE3 control and TJ-PE were tested in the TLR-MCV1 reporter line, and flow cytometry was used to determine the percentage of fluorescent cells. Results were obtained from three independent experiments, shown as mean ± s.d. c Schematics of TJ-pegRNA and targeting strategy for inserting SA-GOI at AAVS1 locus. SA: splice acceptor; GOI: gene of interest. d Bright field and fluorescence images of HEK293T cells 4 days after transfection with PE, TJ-pegRNA, and nicking sgRNA. HEK293T cells transfected with PE plasmid only served as a control (ctrl). Experiments were done two times, and one is shown. Scale bar: 100 µm. e Efficiency of SA-GFP insertion measured by flow cytometry. Results obtained from three independent experiments, shown as mean ± s.d. f Agarose gel of PCR amplicons showing SA-GFP and SA-Puro insertion. Puro: puromycin. The insertion bands of expected sizes are indicated with arrow. The nonspecific bands are indicated with asterisk. Experiments were done two times, and one is shown.