Conditional inactivation of CXCR2 in mice and generation of bone marrow chimeras

We induced homologous recombination in embryonic stem (ES) cells using a construct containing 4.9 kb of gDNA, including exon 1 and part of intron 1–2, an IRES‐LacZ cassette and Neo Cassette were inserted in between Frt sites, a loxP‐site was introduced in 5′ direction to the Neo cassette, and 2.9 kb of gDNA, including CXCR2 exons 2 and 3, was flanked by loxP sites (floxed). The construct was finished by introducing a 3.5‐kb fragment of gDNA serving as 3′ recombination arm (Appendix Fig S3). Correctly targeted ES cell clones were injected into blastocysts to produce a chimeric mouse that transmitted the modified allele through the germ line. A male heterozygous for the targeted allele was bred with a female expressing ubiquitous Flippase (Flp) transgene to ultimately produce animals that had deleted the IRES‐LacZ and Neo cassettes, preserving the loxP sites flanking exons 2 and 3. Those homozygous animals were bred with mice expressing a MRP8‐Cre transgene (Passegue et al, 2004) to ultimately produce animals that had deleted CXCR2 coding exons into neutrophils.

C57BL/6J wild‐type mice (Janvier) underwent lethally whole body irradiation (2 × 5 Gy). Donor bone marrow cells were obtained from Mrp8‐CreCXCR2flox or CXCR2flox mice (litermates). After flushing bones (femur, tibia, humerus), cells were washed, filtered, and intravenously injected (4 × 10⁶ cells/mouse) in sterile saline into recipient mice 1 day after irradiation. Post‐transplantation, recipient mice were reconstituted for another 6 weeks before undergoing LAD occlusion surgical procedure.