Expression in Duodenum of Wild type and Onecut-2 knockout mice at postnatal days 15 and 30

Ablation of the mouse gene for Onecut-2 was reported previously, but characterization of the resulting knockout mice was focused on in utero development, principally embryonic development of liver and pancreas. Here, we examine postnatal development of these Onecut-2 knockout mice, especially the critical period prior to weaning. Microarray technology was used to determine the effect of Onecut-2 ablation on gene expression in duodenum, whose epithelium has among the highest levels of Onecut-2. A subset of intestinally expressed genes showed dramatically altered patterns of expression. Many of these genes encode proteins associated with the epithelial membrane, including many involved in transport and metabolism. Previously, we reported that Onecut-2 was critical to temporal regulation of the adenosine deaminase gene in duodenum. Many of the genes with altered patterns of expression in the Onecut-2 knockout mouse duodenum displayed changes in the timing of gene expression. Duodenal segments were isolated at postnatal days 15 and 30 from wild type 129 mice and 129 mice with the Onecut-2 gene ablated . Total RNA was isolated in biological triplicate for each experimental time point. Each triplicate contained RNA from 5-8 mice of the appropriate age and genotype. Affymetrix microarray technology was used to compare gene expression in wild type duodenum at d15 to gene expression in wild type duodenum at d30 and to compare gene expression in duodenum from Onecut-2 knockout mice at d15 and d30 to each other and to the expression in wild type duodenum.此前已有报道针对小鼠Onecut-2基因进行敲除，但针对该敲除小鼠的表型表征此前主要聚焦于子宫内发育阶段，尤其是肝脏与胰腺的胚胎发育过程。本研究则针对该Onecut-2敲除小鼠的产后发育展开分析，尤其聚焦于断奶前的关键发育期。本研究采用微阵列（microarray）技术，探究Onecut-2敲除对十二指肠基因表达的影响——十二指肠上皮细胞中Onecut-2的表达水平位居前列。部分肠道表达基因的表达模式发生了显著改变，其中多数基因编码与上皮细胞膜相关的蛋白质，涵盖众多参与物质转运与代谢过程的基因。此前本团队曾报道，Onecut-2对十二指肠中腺苷脱氨酶（adenosine deaminase）基因的时序调控至关重要。在Onecut-2敲除小鼠的十二指肠中，表达模式发生改变的基因多呈现出基因表达时序的变化。研究分别于产后第15天与第30天，从野生型129小鼠以及Onecut-2基因敲除的129小鼠体内分离十二指肠节段。每个实验时间点均设置3次生物学重复以提取总RNA，每份重复样本包含5-8只对应年龄与基因型小鼠的RNA。本研究采用Affymetrix微阵列技术，分别对比野生型小鼠产后第15天与第30天十二指肠的基因表达差异、Onecut-2敲除小鼠产后第15天与第30天十二指肠的基因表达差异，以及上述两组敲除小鼠样本与对应时间点野生型小鼠样本的基因表达差异。