Figure 4. Rapid and reproducible antibody reprogramming in PnP-mRuby cells.

(a) Flow cytometry dot plots show PnP-mRuby cells following transfection with CRISPR-Cas9 plasmid and different PnP-sAb donor constructs encoding antibodies specific for HEL or Ebola virus glycoprotein (EBV). The cells were sorted for IgH expression. (b) Flow cytometry dot plots show that all three PnP cell lines express IgH and IgK following sorting in a. (c,d) Similar to a and b are flow cytometry dot plots for PnP-HEL23.2 cells, which were generated in the same manner as PnP-HEL23 with the exception that the Cas9⁺ (2A-BFP) sorting step was omitted. (e) As in Fig. 2f, plot generated from sandwich ELISA demonstrating that all versions of PnP-antibody producing cells secrete IgG into the supernatant. For each sample, three technical replicates were analysed and a four-parameter logistical curve was fitted to the data by nonlinear regression. Data are presented as the mean and error bars indicate standard deviation. (f) As in Fig. 2h, RT–PCR performed on mRNA verified correct insertion and splicing of sAb donors (WT and PnP-mRuby cells received a mixture of forward V_L-specific primers). Throughout this figure, PnP-HyHEL10 corresponds to clone U, PnP-2G4-EBV corresponds to clone AA, and PnP-4G7-EBV corresponds to clone AB, PnP-HEL23.2 corresponds to clone AC (for more details, see Supplementary Table 1).