TI - A Second Tumor with an Insertion at Il2rg . AB - Surprisingly , tumor 7107 contains two Il2rg insertions ( Table 1 ) , which was confirmed by conventional cloning and sequencing ( Figure 2A ) . Thus , 2 of the AKXD murine leukemias with insertions at Lmo2 also contain insertions at Il2rg , a highly significant result ( p = 134x10-9 , see Text S1 for calculation ) . Likewise , tumor 7107 has two insertions at Irs2 (Figure 2B) , another highly significant result ( p = 116x10-5 , see Text S1 for calculation ) . Il2rg and Irs2 are functionally linked in lymphoid cells where it has been shown that Il2rg can promote the PHOSphorylation of Irs2 by binding to and activating the tyrosine kinase Jak3 [18] , which may explain the co-selection for mutations in these genes . To quantitate the Irs2 and Il2rg insertions , we amplified one Lmo2 and one Il2rg insertion using insertion-site -specific primers and real time PCR . The Irs2 insertion was present at about one copy per cell (Figure 2B) , indicating it is present in every tumor cell . The other Irs2 insertion must therefore be in the same tumor cell , either on the same or different chromosome . In contrast , the Il2rg insertion was present at 0.5 copies per cell (Figure 2B) , suggesting it is present in only half the tumor cells of this male mouse . We ruled out hyperploidy for the Irs2 locus and also confirmed that the Il2rg gene was present at one copy per cell ( see Figure S4 ) . The two Irs2 insertions must therefore have occurred first and in the same tumor cell followed by the two Il2rg insertions in different subpopulations of tumor cells . Similar to what was reported previously [9] , Il2rg is not misexpressed in tumor 7107 ( see Figure S3 ) . Most of the genes that were insertionally mutated in this tumor were also highly overexpressed when compared to normal thymus control ( Figure 2C ) . Since our prior study , exons for the Med12 gene were annotated and are shown to direct transcription in the opposite orientation to Il2rg ( see Figure 2A ) . All the tumors have insertions in the same orientation to Med12 except tumor 98-031 and one of the insertions disrupts the second coding exon of Med12 . Med12 was not found to be up-regulated in the tumors and no spliced fusion transcript between provirus and Med12 could be identified ( see Figure S3 ) . It is conceivable that these insertions implicate Med12 in tumorigenesis and not Il2rg , however , the RTCGD contains many common insertion sites in genes of the Il2rg pathway ( eg Il2ra , Il4ra , Il7 , Jak1 , Stat5a/5b ) .