ABSTRACT: Experimental Design Prior studies of C/EBPe-deficient myeloid cells from both humans and mice, demonstrated an essential role for C/EBPe in the normal development and function of both neutrophils and macrophages. There are striking parallels between human neutrophil specific granule disease (SGD) due to mutations in the CEBPE gene and the murine C/EBPe-deficient condition. This indicates that the C/EBPe-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease. In this study, we sought to define further the role that C/EBPe plays in mediating host immune function by identifying possible target genes of C/EBPe in both neutrophils and macrophages. This study was designed to elucidate the effects of the CEBPE gene knockout in murine myeloid cells. Mice and sample preparation The C/EBPE wild type (+/+) and –deficient (-/-) mice (129/SvEv x NIH Black Swiss) were generously provided by K.G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA). They were maintained in pathogen-free facilities on normal chow. To prepare RNA for array hybridization, four age-matched (6-8 weeks) mice of each genotype received an intraperitoneal (IP) injection of 2 ml of 4% sterile thioglycollate broth (Sigma Chemical Co., St Louis, MO). At 24 h post injection, all mice were sacrificed and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice. Total cell numbers were counted and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa stained cytospins (100-200 cells per sample) using a light microscope. The preparations were composed of approximately 30% monocytes, 60% neutrophils and 7-10% lymphocytes in both the C/EBPe+/+ (wt) and C/EBPe-/- (ko) mice. To reduce individual variation in subsequent experiments, the cells either from all wild type or all C/EBPe-null mice were pooled prior to probe synthesis (n=1 pooled RNA for wt and n=1 pooled RNA for ko) Total RNA and protein was isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), Dnase I treated (Promega Corporation, Madison, WI) and purified using an RNeasy spin-column (Qiagen, Valencia, CA). The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel. Hybridization and analysis of the Affymetrix GeneChip Murine 11K Set. The murine 11K set consists of 2 probe arrays (subarray A and B) that allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene (8/96 and Build 4.0) and TIGR (Build 1.0 beta) databases (Affymetrix Inc, Santa Clara, CA). Biotinylated cRNAs were prepared from the pooled RNA samples and fragmented following the manufacturer’s protocol (Affymetrix Inc.). The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.). The probed arrays (n=1) were scanned with a Hewlett Packard Gene Array scanner. The scanned images were analyzed to generate the quantitations based on the images using GeneChip 3.1 software (Affymetrix Inc.). The default settings of the software were used for the analysis. Keywords = CEBPE peritoneal macrophage neutrophil knockout Keywords: other
