Figure 3

Targeting human endogenous loci for GPI-epitope tag knock-in and knockout selection. (a) Double KI of CD5Flag2-bglpA and CD5HA2-bglpA into the human CD59 gene results in selection of highly pure KO cells using double positive cell sorting. 293 T cells were immunostained 3 days post transfection (lower left DotPlot) or after several rounds of Flag⁺ and/or HA⁺ cell sorting (as indicated by arrows) and analyzed for CD59 surface expression (shown on the embedded histogram plots). (b) ELISA quantification of HSA and AFP (control) in the supernatants of HepG2 cells targeted with two epitope tag constructs for the HSA gene; dashed line indicates detection limit, n/d - not detected. Flag+, HA+, db+ and db++ indicate single or double positive cell populations sorted once or twice (see Supplementary Fig. 4 for details). (c) WB analysis of VDAC1 (top image) and VDAC3 (bottom image) in 293 T cells after KI into respective loci and cell sorting using one or two tags (the full-length blots are presented in Supplementary Fig. 8) (d) PCR-amplification of targeted loci from sorted cells (top panel); DNA fragments corresponding to transgene integration generated from db+ cells (blue boxes) were excised and analyzed by Sanger sequencing, whereas DNA fragments with no integration (red boxes) were purified to detect indels using Surveyor assay (bottom gel). (e–g) In-frame strategies (left) and CD5HA2 expression (right) measured in 293 T cells after KI into indicated genes; arrows above the lane indicate active (red) or inactive (gray) start-codons in-frame with transgene; arrows below the lane are AUG-codons out-of-frame with CD5HA2; dashed line indicates the codon that has A/G at + 3 position and can initiate translation of short peptides; translated products as fusions of VDACs LD, Ku70 vWa (von Willebrand A-like domain) or KPNA1 IBB (Importin beta binding domain) with transgene are shown as rectangle boxes. Images in a, c, and d are representative of at least two experiments. The averaged results from three independent experiments with standard deviations are presented in b and e-g. * and **, values are different by Student’s t-test at p < 0.05 and p < 0.01, respectively.