CEBPE WT vs. KO peritoneal lavage cells. Mus musculus

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

实验设计
此前针对人类与小鼠的C/EBPe缺陷型髓系细胞开展的研究已证实，CCAAT增强子结合蛋白ε（C/EBPe）在中性粒细胞与巨噬细胞的正常发育及功能发挥中具有关键作用。因CEBPE基因突变引发的人类中性粒细胞特异性颗粒病（SGD）与小鼠C/EBPe缺陷模型之间存在显著相似性，这表明C/EBPe缺陷型小鼠模型可作为强有力的工具，用于进一步解析这种罕见的人类疾病。本研究旨在通过鉴定中性粒细胞与巨噬细胞中C/EBPe的潜在靶基因，进一步阐明C/EBPe在介导宿主免疫功能中发挥的作用，本研究的设计目标为解析CEBPE基因敲除对小鼠髓系细胞的影响。

实验动物与样本制备
C/EBPe野生型（+/+）与缺陷型（-/-）小鼠（129/SvEv × NIH黑瑞士小鼠）由美国加利福尼亚州圣地亚哥市Anadys制药公司的K.G. Xanthopoulos以及马萨诸塞州剑桥市千禧制药公司的Julie Lekstrom-Himes惠赠。所有小鼠均饲养于无特定病原体（SPF）级动物房，饲喂普通饲料。为制备用于芯片杂交的RNA，每组基因型选取4只6~8周龄的同龄小鼠，经腹腔（IP）注射2 mL 4%无菌硫乙醇酸盐肉汤（美国密苏里州圣路易斯市Sigma化学公司）。注射后24小时处死所有小鼠，采用Hanks平衡盐溶液（HBSS；Sigma化学公司）灌洗收集腹腔渗出细胞，并置于冰上保存。通过计数总细胞数，并使用光学显微镜对瑞氏-吉姆萨染色的细胞涂片（每份样本计数100~200个细胞）进行分类计数，以测定中性粒细胞与巨噬细胞的占比。在C/EBPe野生型（wt）与缺陷型（ko）小鼠的样本中，细胞组成均约为30%单核细胞、60%中性粒细胞以及7%~10%淋巴细胞。为降低后续实验中的个体差异，在探针合成前将所有野生型小鼠或所有C/EBPe缺陷型小鼠的细胞分别混合（野生型组与缺陷型组各制备1份混合RNA样本，n=1）。采用TRIzol试剂（美国马里兰州盖瑟斯堡市Life Technologies公司）按照厂商说明书从细胞中提取总RNA与总蛋白，经脱氧核糖核酸酶I（Dnase I，美国威斯康星州麦迪逊市Promega公司）处理后，使用RNeasy离心柱（美国加利福尼亚州瓦伦西亚市Qiagen公司）进行纯化。通过变性琼脂糖凝胶电泳检测RNA样本的质量与完整性。

Affymetrix小鼠11K基因芯片的杂交与数据分析
小鼠11K芯片包含2张探针阵列（子阵列A与B），可用于检测源自UniGene（8/96版本及Build 4.0）与TIGR（Build 1.0 beta版本）数据库的11000余个基因的相对表达丰度（美国加利福尼亚州圣克拉拉市Affymetrix公司）。按照Affymetrix公司的试剂盒说明书，从混合RNA样本中制备生物素标记的互补RNA（cRNA）并进行片段化处理。使用GeneChip流体工作站400（Affymetrix公司），按照厂商说明书对小鼠11K A与B阵列进行预杂交与杂交反应。使用惠普基因阵列扫描仪对探针杂交后的阵列（n=1）进行扫描。使用GeneChip 3.1软件（Affymetrix公司）对扫描得到的图像进行分析，生成基因表达定量结果，数据分析采用该软件的默认参数设置。

关键词：CEBPE、腹腔巨噬细胞、中性粒细胞、基因敲除；其他关键词：无