Figure 1.

A Bxb1 recombinase-based platform for transgene library expression. (A) Disruption of the AAVS1 locus on chromosome 19 stimulates genomic landing pad insertion through homology directed repair. (B) The genomically integrated landing pad is subsequently used to efficiently integrate a transgenic cassette using the site-specific Bxb1 recombinase. (C) Depletion of BFP+ cells over time after transfection of the landing pad plasmid shown in panel B, as determined by flow cytometry. Cells were considered BFP+ if their BFP fluorescence was greater than that of untransfected cells. Loss of BFP signal over time is largely reflective of dilution or degradation of transfected plasmid. Error bars reflect standard error (N = 3). (D) After three weeks of post-transfection growth with additional sorts for BFP+ cells on days 5 and 13, BFP fluorescence was measured by flow cytometry. Cells within the box are likely to contain genomic landing pad insertions. Color indicates point density from low (blue) to high (red).