Fig. 3.

Generation of genomic safe harbor lines for stable expression of protein biosensors. (A) A schematic showing the mechanism for generating the reporter line using Genomic Safe Harbor-RMCE (Recombinase Mediated Cassette Exchange) approach. Abbreviations: TALEN-Transcription activator-like effector nucleases (Left and Right), AAVS1-Adeno-Associated Virus Integration Site 1 (LA-Left Arm, RA-Right Arm). (B) Validation of LoxP-CAG-GFP (ZYP037) insert into AAVS1 genomic locus using junction PCR. Amplicon of the expected size validating the right and left junction are shown. (C) Characterization of genomic safe harbor lines D149-GSH and LSPH004-GSH: Expression of the NSC markers Nestin and SOX2 detected by immunocytochemistry. GFP expression from the safe harbor construct marker is also shown. Scale bars: 40 μm. (D) Representative confocal image (maximum z-projection) showing expression and localization of mCherry::PH-PLCδ (magenta) and cytosolic GFP (green) in D149 PIP_2R reporter line colonies. Nucleus stained using Hoechst (cyan). Scale bars: 50 μm. (E) Representative flow cytometry data to illustrate the gating strategy for FACS purification of (Ei) GFP positive cells for genomic safe harbor lines, to select for healthy GFP positive cells, side scatter (SSC-A, log, Y-axis) and signal from the excitation of cells with the 488-nm laser (GFP-A, log, X-axis) are plotted. (Eii) mCherry positive cells for PIP₂ reporter lines were selected for by plotting signal from the excitation of cells with the 568-nm laser (mCherry-A, log, Y-axis) against signal from the excitation of cells with the 488-nm laser (GFP-A, log, X-axis). (F) Western blot showing OCRL (detected using the OCRL antibody), mCherry::PH-PLCδ (detected using the mCherry antibody) in NSC PIP_2R reporter lines. GAPDH was used as a loading control.