Focal expression of Irga6 is dependent on interferon (IFN)-pathway. (A) Focal expression of Irga6 is eliminated in mice deficient in the IFNγ-response pathway. High Irga6 expression foci were counted in livers and kidneys from two mice deficient in IFN-pathway components and from two appropriate control wild-type (WT) mice as described (see Methods section). Focus numbers from knockout (KO) organs are presented as a percentage of the focus numbers in control WT mice (ko/wt). For raw data see Additional file 1: Table S1. Kidneys of RAG-/- and JHT strain could not be analysed (ND), because of generally elevated Irga6 expression (Additional file 2: Figure S1). (B) Irga6 focal expression is not triggered by immune responses to microbial components. Irga6 focal expression was assayed in organs from mice deficient in TLR2/4 and TLR9, MyD88 and germ-free (GF) mice. GF mice came from three different sources [GF(P) Portugal, GF(CH) Switzerland, GF(S) Sweden]. (C) IFNγ-inducible transcripts of Irga6 (1A) dominate focal expression in liver. IFNγ-inducible (Irga6-1A) and liver-specific (Irga6-1B) transcripts were quantitated by quantitative real-time polymerase chain reaction (RT-PCR) from focal (F) and non-focal (NF) material isolated from stained tissue sections by laser microdissection (top left: left panel, liver section before microdissection of cored patch; middle panel, after microdissection; right panel, microdissected patch). Irga6-1A and Irga6-1B transcripts were amplified using selective primers for the 1A and 1B 5'-exons and a common 3' primer in the coding exon (top right). The ratio of expression of 1A to 1B is given for F and for NF (bottom left) as the mean of two independent experiments (error bars indicate standard deviations). Twenty clones amplified for Irga6-1A from both F and NF were sequenced. The dominance of Irga6-1A in F is very high since only 5.9% of clones amplified for 1A from NF liver were specific for Irga6-1A, compared with 100% from F (see Methods section). (D) IFN-γ is expressed in liver cored patches. Total liver tissues (liver), Irga6 liver patches (P) and CP were collected as described (C). RT-PCR was carried out using IFN-γ specific primers and the products run in an agarose gel. GAPDH was used as control.