Lysm cre transgenic mice

Wild-type C57BL/6 (Cat#: SM-001), Mettl14^flox/flox (Cat#: NM-CKO-190007), and Mettl3^flox/flox (Cat#: NM-CKO-190006) mice bearing LoxP sites were from Shanghai Model Organisms Center. LysM-Cre transgenic mice (Cat#: 004781) were from Jackson Laboratory. Mettl14^flox/flox/Mettl3^flox/flox mice and LysM-Cre transgenic mice were crossed to generate the conditional gene knockout animals. All mice were of the C57BL/6 background, aged 6–8 weeks, and were used in this study irrespective of sex. The mice were housed at 25 °C with a 12 h/12 h light/dark cycle and ~50% humidity. The experimental and control mice were bred separately under specific pathogen-free conditions. For experiments, the mice were euthanized in a CO₂ chamber. The protocols related to animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanxi Medical University. Both male and female mice were used as our preliminary data did not show sex as a confounding factor.

Animals were used according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with the protocol approved by the Institutional Animal Care and Use Committee at the Augusta University. The floxed Adora2a (Adora2a^flox/flox) mice were provided by Dr Joel Linden (La Jolla Institute for Allergy and Immunology, La Jolla, California, USA). Cell-specific inactivation of Adora2a in ECs or in macrophages was achieved by cross-breeding Adora2a^flox/flox mice with Cdh5-Cre transgenic mice (The Jackson Laboratory, Stk#006137, Bar Harbor, ME) or Lysm-cre transgenic mice (The Jackson Laboratory, Stk#004781), respectively. Global homozygous Adora2a (Adora2a^−/−) knockout mice were generated as previously described^{54 (link)}. All mice were on a C57BL/6J background.

We generated mice harboring a floxed allele of Rora (Rora^fl/fl) by flanking exon 3 with two loxP sequences. The strategy and outcome on RORα1 and RORα4 are summarized in figures S1A and S1B respectively. Briefly, a targeting vector containing loxP sites, a FRT-floxed neomycin cassette and homologous regions surrounding exon 3 was constructed and transfected in embryonic stem (ES) cells derived from 129/Sv. After screening for homologous recombination by southern blot and PCR, ES cells were injected into C57BL/6 blastocysts. Neomycin cassette was removed in vivo by using FLP deleter mice in C57BL/6 background. Finally, mice were backcrossed with C57BL/6 J mice for at least six generations.
To achieve the deletion of RORα in macrophages, Rora^fl/fl mice were crossed with LysM-Cre transgenic mice (Jackson laboratory) in which Cre recombinase is expressed under the control of endogenous Lyz2 promoter. Littermates RORα-deficient (MKO, Rora^fl/fl Lyz2^Cre/+) and WT (Rora^+/+Lyz2^Cre/+) mice were generated by crossing Rora^fl/+Lyz2^Cre/Cre with Rora^fl/+Lyz2^+/+ mice. All mice were genotyped twice.

C57BL/6 and neutrophil elastase (Ne^−/−) mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME), and bred at Case Western Reserve University and UC Irvine. Gsdmd^−/− mice were generated by Dr. Russell Vance (University of California, Berkeley) on a C57BL/6 background as described^{51 (link)}, and were bred at UC Irvine. Myeloid specific deletion of Atg7 (Atg7^MΔ) was obtained by breeding Atg7^f/f mice (parent strain) with LysM-Cre transgenic mice (from Jackson Laboratory) as described^{27 (link)}, and bones were sent to us by Dr. Tony Eissa (Baylor College of Medicine, Texas). All animals were housed in pathogen free conditions in microisolator cages and were treated according to institutional guidelines following approval by the Case Western Reserve University and the University of California IACUC.

All animal experiments were carried out according to the procedures approved by the Institutional Review Board and the Regierungspräsidium Tübingen and comply with all relevant ethical regulations regarding animal research. RGM-A^+/- and littermate control mice were bred and genotyped using the following primers: wild type; 5′- CAG GTA GGC ACA ACT CCT TGG TGG-3′, 5′- TTA GCA CGT CTG AGC CTG TGT CCG-3′; knock-out: 5′- TGC GAA GTG GAC CTG GGA CCG CG-3′, 5′- CAT CCA ACA AGG CTC CAC TGG AAG G-3′^{7 (link)}. Floxed RGM-A mice were bred with LysM-Cre transgenic mice (The Jackson Laboratory) to generate myeloid cell lineage-specific knockout animals. LysM-Cre– floxed littermates were used as their control. 12/15-LOX-deficient mice (12/15-LOX^−/−; The Jackson Laboratory, # 002778) and littermate control mice were bred and genotyped using following primers: mutant: 5′- GGG AGG ATT GGG AAG ACA AT-3′, common: 5′- GGC TGC CTG AAG AGG TAC AG-3′, wild type: 5′- CCA TAG ACG AGA CCA GCA CA-3’^{41 (link)}.