Targeting of the Gimap4 locus. (A) An internal 1.1 kb EcoRV fragment (comprising the 3′ end of the intron and most of exon 3 encoding the GTPase domain) was replaced by a blunted 1.4 kb BamHI/KpnI selection cassette containing a floxed neomycin gene. (B) Gimap4-targeted mice were screened by Southern blot of EcoRI-digested tail DNA using a 3′-specific probe and (C) by PCR; a 1.35 kb and a 1.65 kb band identified the wt and the targeted allele, respectively. (D) Targeted Gimap4 ko's were also crossed with Cre deleter, and PCR analysis was performed; the wt allele is 1.35 kb, the ko allele 0.4 kb. (E) Total RNA from DNIII, DNIV, ISP, and DP thymic subsets, obtained from Rag-deficient mice triggered with or without anti-CD3, as well as RNA from peripheral T cells (mT) have been used for quantitative PCR for Gimap family members. GAPDH was used as an endogenous control for normalization of targets.