Deregulated expression programs in the hypomorphic Dnmt3b mutant mice

The function of Dnmt3b, of which deregulated activity is linked to several human pathologies, was studied using Dnmt3b hypomorphic mutant mice with reduced catalytic activity. Microarray analysis of deregulated expression programs in the hypomorphic Dnmt3b mutant mice (m3/m24) was combined to an analysis of the molecular mechanisms involved in the illegitimate activation of a specific set of genes. Mouse embryonic fibroblasts and thymus tissues isolated from 12.5 dpc and 18.5 dpc, respectively, wild-type and hypomorphic embryos (129SvJae x C57BL/6 hybrid genetic background). Total RNA was isolated using RNeasy Mini kit (QIAGEN), amplified, labeled, and hybridized following a previously described protocol (Le Brigand et al., Nuc Acid Res, 2006). Total RNA was coupled with Cyanine 3 or 5 and then hybridized in competition with reference RNA composed of a pool of total RNA isolated from wild-type thymus or MEF cells (thymus, n=5; MEF, n=5); two dye-swap were realized leading to the analysis of 18 microarrays for thymus (2 microarrays were excluded for unsufficient quality) and 20 for MEF cells. Arrays were then scanned with Agilent G2565AA Microarray Scanner (Agilent Technologies).DNA甲基转移酶3b (Dnmt3b)的活性失调与多种人类疾病相关，本研究通过构建催化活性降低的Dnmt3b低功能突变小鼠，对其功能展开探究。本研究针对该低功能Dnmt3b突变小鼠（m3/m24）中表达程序失调情况开展微阵列（Microarray）分析，并结合特定基因集异常激活所涉及的分子机制解析。实验样本中，小鼠胚胎成纤维细胞（Mouse embryonic fibroblasts, MEF）取自妊娠12.5天（days post coitum, dpc）的胚胎，胸腺组织则取自妊娠18.5天（days post coitum, dpc）的胚胎；所有胚胎均包含野生型与低功能突变型个体，且均为129SvJae与C57BL/6的杂交遗传背景。总RNA提取采用RNeasy Mini试剂盒（QIAGEN），并依照已发表的实验方案（Le Brigand等，《核酸研究》，2006年）完成扩增、标记与杂交步骤。将总RNA与花青苷3（Cy3）或花青苷5（Cy5）偶联后，与由野生型胸腺或MEF细胞总RNA混合制备的参考RNA进行竞争性杂交（胸腺样本n=5；MEF样本n=5）；通过两次染料交换实验，最终共分析18张胸腺样本微阵列（其中2张因质量不达标被排除）与20张MEF细胞样本微阵列。随后使用安捷伦G2565AA微阵列扫描仪（Agilent Technologies）对芯片进行扫描。