Genetic Mouse Models for RIPK1 and Immune Signaling

Ripk1^fl/fl, Ripk1^D138N/D138N (ref. 29 ), Trif^fl/fl, Fadd^fl/fl (ref. 30 (link)), FADD-IRES-eGFP^fl/fl (ref. 4 (link)) and R26-Stop^flIkk2ca^{22 (link)} mice expressing a constitutively active Ikk2 (Ikk2ca) transgene were generated by gene targeting in C57BL/6 embryonic stem (ES) cells. FLPe-Deleter^{31 (link)} mice were used to delete the FRT-flanked neo cassette and Cre-Deleter^{32 (link)} mice were employed to delete the RIPK1 floxed sequences in the germ line and generate Ripk1^−/− mice and MEFs. Villin-Cre^{33 (link)}, VillinCreER^T2 (ref. 34 (link)), K14-Cre^{35 (link)}, Tnfr1^−/− (ref. 36 (link)), Myd88^−/− (ref. 37 (link)) and Ripk3^−/− (ref. 38 (link)) mice were backcrossed for at least ten generations into the C57BL/6 genetic background. Mice were maintained at the SPF animal facilities of the Institute for Genetics, University of Cologne, and of the University of Massachusetts Medical School and kept under a 12 h light cycle, and given a regular chow diet (Harlan diet no. 2918 or Prolab Isopro RMH3000 5P76) ad libitum. Germ-free mice were produced at the gnotobiotic facilities of the University of Ulm and of the Hannover Medical School. All animal procedures were conducted in accordance with European, national and institutional guidelines and protocols were approved by local government authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany). Animals requiring medical attention were provided with appropriate care and excluded from the experiments described. No other exclusion criteria existed. For antibiotic treatment of RIPK1^IEC-KO mice one of two different broad spectrum antibiotic mixtures was added to the drinking water starting from embryonic day (E)17.5: 1 g l⁻¹ ampicillin (ICN Biomedicals), 1 g l⁻¹ neomycin (Sigma), 0.5 g l⁻¹ Meronem (AstraZeneca) and 0.5 g l⁻¹ ciprofloxacin (Fluka) or 1 g l⁻¹ ampicillin (ICN Biomedicals), 1 g l⁻¹ metronidazole (Sigma), 0.5 g l⁻¹ vancomycin (Eberth) and 0.2 g l⁻¹ ciprofloxacin (Fluka). In VillinCreER^T2/Ripk1^fl/fl mice a modified version of the first protocol was employed, where Meronem was replaced by 0.5 g l⁻¹ vancomycin (Eberth). As neither the overall physiological response nor tissue pathology differed between the two regimens, mouse groups receiving either antibiotic mixture were analysed together and data from individual experiments were pooled. VillinCreER^T2 recombinase activity was induced by three daily intraperitoneal administrations of 1 mg tamoxifen. Littermates not carrying the Villin-Cre, Villin-CreER^T2 and K14-Cre transgenes were used as controls in all experiments. Mice of the indicated genotype were assigned at random to groups. Mouse studies were performed in a blinded fashion. Unless otherwise indicated, mice were analysed at 3 weeks of age. Groups included male and female animals.