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. 2017 Oct 20;45(20):11837–11857. doi: 10.1093/nar/gkx847

Figure 1.

Figure 1.

rAAV-mediated generation of FANCD2−/− (D2−/−), FANCI−/− (I−/−) and FANCI−/−:FANCD2−/- (ID2 DKO) cell lines. (A) Schematic of FANCD2 (left panel) and FANCI (right panel) targeting strategies in HCT116 cells. FANCD2 exon 12 and FANCI exon 10 were targeted for deletion by rAAV gene targeting. The first allele targeting was performed using conditional rAAV vectors for FANCD2 and FANCI. The conditional vectors contain rAAV inverted terminal repeats (ITRs) (gray boxes), homology arms (white boxes), an NEO selection cassette (green boxes) and the targeted exon (blue boxes) flanked by LoxP sites (orange triangles). Targeted clones were treated with Cre recombinase to remove the NEO selection cassette. (B) The second allele targeting was performed in the FANCD2flox/+ cell line (f/+) using a FANCD2 knockout rAAV vector and in the FANCIflox/+ cell line (f/+) with the same FANCI conditional rAAV vector utilized in the first round of targeting. Targeted clones were treated with Cre recombinase to remove both the NEO selection cassette and the conditionally floxed exons, resulting in the generation of FANCD2−/− (D2−/−) and FANCI−/− (I−/−) cell lines.