Microarray screening of RNA samples from the olfactory epithelium from wild type and beta3GnT1 mutant mice

Dr. Schwarting's research is focused on the analysis of developmentally regulated cell surface molecules and their role in axon guidance and neuronal migration, using the olfactory system as a model. The interaction of cell surface glycans with endogenous lectins in the extracellular matrix provides one mechanism by which axons can utilize specific pathways as they grow towards their targets. Beta3GnT1 mutant mice lose lactosamine expression and have an olfactory axon guidance phenotype (Henion et al, 2005). Olfactory neurons are capable of regeneration and we know that when these neurons regenerate in beta3GnT1 mutant they synthesize lactosamine by a secondary mechanism. By comparing RNA from the olfactory epithelium from wild type and mutant mice, we would expect to see the upregulation of glycosyltransferases that could produce lactosamine in the absence of beta3GnT1. In a preliminary experiment carried out by the CFG staff, we saw that radical fringe, a beta 3GnT, was upregulated in the olfactory epithelium, although it is probably not responsible for the new lactosamine expression. beta3GnT1 Knock out. Strain info: The b3GnT1 mice were generated on a mixed 129Ola-C57BL/6 background and backcrossed to C57BL/6 for four generations. They were then crossed to I7-Internal ribosomal entry site (IRES)-tau greeen fluorescent protein (GFP) mice generated by Dr. Peter Mombaerts. RNA preparations from wild type and beta3GnT1 mutant mouse olfactory epithelium were sent to Microarray Core (E). Three replicate samples from each condition were used in the study. The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays. Data was analyzed to determine glycosyltransferase expression changes in beta3GnT1 mutant mice, with specific interest on glycosyltransferases that could produce lactosamine in the in the absence of beta3GnT1.施瓦廷（Schwarting）博士的研究聚焦于发育调控型细胞表面分子的解析，及其在轴突导向与神经元迁移中的功能，并以嗅觉系统作为研究模型。细胞表面聚糖与细胞外基质中的内源性凝集素之间的相互作用，为轴突在向靶标生长过程中利用特定通路提供了一种潜在机制。β3GnT1（β1,3-N-乙酰葡糖胺基转移酶1）突变小鼠会丧失乳糖胺表达，并出现嗅觉轴突导向异常表型（Henion等，2005）。嗅觉神经元具备再生能力，我们已知当此类神经元在β3GnT1突变体中再生时，可通过次级合成途径产生乳糖胺。通过对比野生型与β3GnT1突变型小鼠嗅觉上皮的转录组RNA，我们预期能够观察到在缺失β3GnT1的情况下，可合成乳糖胺的糖基转移酶的上调表达。在由CFG团队开展的预实验中，我们观察到作为β3GnT家族成员的边缘素（Radical Fringe）在嗅觉上皮中出现上调表达，尽管其大概率并非介导新生乳糖胺表达的关键因子。β3GnT1敲除小鼠品系信息：该b3GnT1小鼠以混合129Ola-C57BL/6遗传背景为基础构建，并回交至C57BL/6品系共计4代；随后将其与由彼得·蒙巴茨（Peter Mombaerts）博士构建的I7-内部核糖体进入位点（IRES）-tau绿色荧光蛋白（GFP）小鼠进行杂交。从野生型与β3GnT1突变型小鼠的嗅觉上皮中提取的RNA被送至微阵列核心实验室（E）。本研究采用了每种实验条件下的三份重复样本。对RNA进行扩增、标记，并与GLYCOv3微阵列进行杂交。对所得数据进行分析，以鉴定β3GnT1突变型小鼠中糖基转移酶的表达变化，重点关注可在缺失β3GnT1的条件下合成乳糖胺的糖基转移酶。