Fig. 1.

Scheme for functional inactivation of the human Ku86 locus. (A) A diagram of a partial Ku86 genomic locus. Exons are shown (not to scale) as numbered rectangles. In addition, a diagram of the rAAV targeting vector is shown. The filled triangles represent LoxP sites, and the hatched rectangle represents the NEO gene. (B) Diagram of the Ku86^NEO/+ cell line generated by the first round of targeting. (C) Ku86^NEO/+ cells were transiently transfected with a Cre expression plasmid (+pGK-Cre), and a clone of cells (Ku86^flox/+) that had lost the NEO gene but retained the floxed exon 3 was identified. (D) Ku86^flox/+ cells were then subjected to a second round of targeting using an rAAV exon 3 knockout vector. This generated the Ku86^flox/NEO cell line. (E) The Ku86^flox/NEO cell line was then infected with AdCre (+AdCre), and a clone of cells that were G418 sensitive (Ku86^flox/−) was isolated. (F) These Ku86^flox/− cells can be converted into Ku86 null (Ku86^−/−) cells at the investigator's discretion by infecting them with AdCre.