Fig. 7: Two functional scFv variants can be generated with recombination and displayed.
a Diagram of the pY110-CJCS-PG-H20 plasmid. AGA2 and a VH fragment are flanked by a 12-RSS followed by an intersignal region and then two modules each consisting of a 23-RSS, a gene fragment that contains a unique VHCDR3, and a terminator. Triangles represent RSSs. RAG-mediated recombination creates either an anti-PSCA or anti-GPC3 product which can be displayed on the surface of the cell. b Demonstration of expected phenotype change during RAG-mediated recombination of the split-scFv substrate. Initially, the cells cannot display a functional scFv. Depending on the outcome, a fraction of cells will display either an anti-PSCA or anti-GPC3 antibody after recombination. c scFv recombination results measured via flow cytometry. Wild-type EBY100 cells were compared to the recombination strain which expresses NLS-RAG1core, RAG2, and HMGB1. Cells were induced for 8 d in SG-Trp (Galactose) or SD-Trp (Glucose), then grown for 1 d in SD-Trp followed by 1 d in buffered SG-Trp. Due to fluorophore overlap, the cells were split into two separate staining reactions: one for PSCA and Myc and another for GPC3 and FLAG. In (c) data are presented as mean values ± SD; n = 3 biological replicates. Statistical significance was calculated with a two-way ANOVA with repeated measures to compare scFv display and Tukey test (****p < 0.0001). Source data are provided in the Source Data file.
