Resistin-Induced Inflammation in Mice

Human resistin transgenic mice (hRetnTg⁺) were generated as previously described by Mitchell Lazar [26] (link). Briefly, the human resistin gene, along with 21,300 bp upstream and 4,248 bp downstream of the human resistin start site, were engineered through a bacterial artificial chromosome. hRetnTg⁻ mice were backcrossed onto WT C57BL/6 mice bred in house to generate mRetn^−/− mice. In the resistin binding assay, CX3CR1^GFPPGRP^dsred mice were used as reporters for monocytes and neutrophils respectively [58] (link). Recombinant human resistin (Peprotech) was used for in vivo intraperitoneal injections (500 ng/mouse at days -1 and/or day -3). In some experiments, mice were also treated intratracheally with 500 ng hResistin. Cells from the peritoneal cavity were recovered at 24, 48, and 72 hours and analyzed by flow cytometry and real-time PCR. Recombinant hResistin was tested for endotoxin contamination using the Pierce Limulus Amebocyte Lysate assay (Thermo Scientific) according to manufacturer's instructions under sterile conditions. For controls, mice were injected i.p. with PBS or the limit of detection concentration of LPS (1.5pg/mL). All animals in the experiment were age-matched (6–10 week old) and gender-matched, and housed five per cage under ambient temperature with 12 hour light/dark cycle.