Transcription profiling by array of spleens from mice with Nix knocked out

Normal erythropoiesis requires a critical balance between proapoptotic and antipaoptotic pathways. Bcl-xl, an antiapoptotic protein is induced at end-stages of differentiation of erythroid precursors in response to erythropoietin. The details of the proapoptotic pathway and the critical proapoptotic proteins inhibited by Bcl-xl in erythropoiesis are not well understood. We employed gene targeting to ablate Nix, a proapoptotic BH3-domain only Bcl2 family protein, which is known to be transcriptionally induced during erythropoiesis. Nix null mice exhibited reticulocytosis and thrombocytosis in the peripheral blood; and profound splenomegaly with erythroblastosis in the spleen and bone marrow despite normal erythropoietin levels and blood oxygen tension. In vivo apoptosis was diminished in erythroblast precursors from Nix null spleens. To define the molecular consequences of Nix ablation on apoptosis and erythropoiesis, we conducted a detailed comparative analysis of gene expression in spleens from 8 week old Nix null mice and wild type controls. Of 45,101 genes analyzed, 514 were significantly upregulated and 386 down-regulated in Nix-/- splenocytes. Functional cluster analysis delineated the ten most highly regulated gene sets, revealing increased levels of cell cycle and erythroid genes, with decreased levels of cell death and B-cell genes. Experiment Overall Design: In the study, we hybridized RNA from 8 week old spleens of wild type (WT) control and Nix null mice (Nix-/-) to Affymetrix MOE430A GeneChip arrays containing 45,101 well characterized mouse genes/ESTs.正常红细胞生成（erythropoiesis）需要促凋亡与抗凋亡通路之间维持关键平衡。Bcl-xl作为一种抗凋亡蛋白，可在红细胞前体分化的终末阶段，响应促红细胞生成素的信号而被诱导表达。目前，关于红细胞生成过程中的促凋亡通路细节，以及Bcl-xl所抑制的关键促凋亡蛋白，尚未得到充分阐明。本研究采用基因靶向技术敲除Nix——一种仅含BH3结构域的促凋亡Bcl-2家族蛋白，已知该蛋白在红细胞生成过程中会被转录诱导。Nix基因敲除小鼠的外周血可观察到网状红细胞增多症（reticulocytosis）与血小板增多症（thrombocytosis）；尽管其促红细胞生成素水平与血氧张力均处于正常范围，但脾脏与骨髓中却出现显著的脾肿大（splenomegaly），并伴随幼红细胞增多症（erythroblastosis）。对来自Nix敲除小鼠脾脏的红细胞前体进行检测，发现其体内凋亡水平显著降低。为阐明Nix敲除对细胞凋亡及红细胞生成的分子调控效应，我们对8周龄Nix敲除小鼠与野生型（wild type, WT）对照小鼠的脾脏组织开展了详尽的基因表达比较分析。在本次分析涉及的45101个基因中，Nix-/-脾细胞内有514个基因显著上调，386个基因显著下调。功能聚类分析筛选出调控程度最高的10个基因集，结果显示细胞周期相关基因与红细胞系相关基因的表达水平升高，而细胞死亡相关基因与B细胞相关基因的表达水平降低。实验整体设计：本研究将8周龄野生型对照小鼠与Nix敲除（Nix-/-）小鼠的脾脏RNA，与搭载45101个经过充分注释的小鼠基因/表达序列标签（expressed sequence tags, ESTs）的Affymetrix MOE430A基因芯片（GeneChip array）进行杂交实验。