Generation of γ_c-cKO mice. (A) Targeting strategy to generate γ_c-cKO allele. Numbered boxes = exons, gray boxes = out of frame exons as a result of Cre-mediated deletion. Neo^R = neomycin resistance cassette. Black triangles = loxP sites. Gray ovals = frt sites. K, S = Kpn1 and Sac1 restriction sites, respectively. Locations of PCR primers are indicated with facing arrows. Location of 3′ probe for Southern blot is indicated. Configuration of the γ_c germline KO allele used in this study is shown for comparison. (B) PCR confirmation of proper integration of the 5′ end of construct in ES cells and genomic DNA from female knockin offspring. PCR amplification of a 2.9 Kb fragment flanking exon 1 and loxP integration site followed by Sac1 restriction digest reveals a 315 bp WT band and 365 bp targeted band as indicated in A. ES cells have only one targeted band because they are derived from male origin and thus have only one X chromosome. (C) Southern blot confirmation of proper integration of 3′ end of construct. Kpn1 restriction digest results in 9.2 Kb WT fragment and 3.2 Kb targeted fragment as depicted in A. (D) Confirmation that after Actin-flp recombinase-mediated deletion of the Neo^R cassette, expression of the γ_c^fl allele is equal to WT γ_c expression in the absence of Cre throughout T cell development.