GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses
Fig 1
Generation of Gpr108-null mice.
(A) Schematic diagram of construction of Gpr108 knockout mice. The murine Gpr108 locus contains 17 exons. Using a targeting BAC bearing the selection marker tk-2A-neo flanked by R4 integrase attB and attP sites, exons 1 to 17 were deleted, yielding the MT1 ES cell line. After expression of R4 integrase in MT1 cells, the resistance elements were removed as shown in MT2. The probes used for MLPA, 5wt, 3wt, 5m, 3m, neo, C1 and C2 are indicated. The primers P1/P2, P3/P4 used for PCR genotyping and P5/P6 for R4 deletion test are also shown. (B) Representative MLPA fragment analysis of 5wt, 3wt, 5m, 3m, neo, C1, C2 for different target events in wild-type cells as well as MT1 and MT2 targeted ES cells. Using HP1 (C1) and ITGB3 (C2) as internal controls to normalize intensities, the 5wt and 3wt signal intensities were observed to be diminished by approximately one half in correctly targeted MT1 cells compared to those in wt cells and the copy numbers of 5m, 3m and neo were identified as single copy or greater by analysis of all ES clones exhibiting resistance to G-418. In MT2 ES cells, the neo MLPA signal was not observed following excision by R4 integrase. (C) Mouse genotypes by PCR of wild-type (WT), heterozygous (Het) and homozygous (KO) mice are indicated. (D) Gpr108 mRNA abundance in WT, Het and KO MEF cells. Gpr108 mRNA was absent from KO MEF cells and present in Het at approximately 50% of the level found in wild-type MEF cells. (E) Gpr108 mRNA detection in spleen and lung of WT and KO mice. Gpr108 mRNA was absent in tissues from KO mice. (F). mRNA transcriptome panel of immune response and immediate early genes (IEGs) in BMDM cells derived from Gpr108+/+ (n = 3) and Gpr108-/- (n = 3) mice in the absence or presence of different TLR agonists. Each column presents the mRNA expression of BMDM cells without or with different treatments. The intensity represents the magnitude of the difference. Red and green denote low and high expression, respectively.