Transcription profiling of mouse developing kidney to investigate Wnt4-dependent gene expression (GUDMAP Series_id: 13)

Our laboratory's interest is in understanding the molecular principles that underlie the regional organization of the mammalian metanephric kidney. Our goal is to generate a detailed spatial map of the cellular expression of selected regulatory genes during mammalian kidney development. The goal of this study is to identify a population of genes that are enriched in the renal vesicle (RV) and its derivatives using Wnt4 mutants. Experiment Overall Design: Wnt4 is required for renal vesicle (RV) induction. Therefore, RV and the derivatives (s-shaped body and eventually the mature nephron) are missing in Wnt4 mutants. At E14.5, these structures are present in wildtype kidneys. Transcriptional profile comparison between E14.5 wildtype and Wnt4 mutants therefore identify genes expressed in the RV and derivatives. Minimally pooled kidney samples were used as single biological replicates. Total RNA was isolated from these pools and subjected to a single round of amplification for use on Affymetrix arrays.我们实验室的研究兴趣在于解析介导哺乳动物后肾（metanephric kidney）区域化构建的分子机制。本研究的目标是构建哺乳动物肾脏发育过程中特定调控基因的细胞表达精细空间图谱。本次实验的核心目的为借助Wnt4突变体，筛选出在肾小泡（renal vesicle, RV）及其衍生结构中富集的基因群。 实验整体设计： Wnt4是肾小泡诱导过程的必需调控因子，因此Wnt4突变体无法形成肾小泡及其衍生结构（S形小体，最终发育为成熟肾单位）。在胚胎期14.5天（E14.5），野生型小鼠肾脏中存在上述结构。因此，对比胚胎期14.5天野生型与Wnt4突变体小鼠肾脏的转录组谱，可筛选出在肾小泡及其衍生结构中特异性表达的基因。 本实验采用少量混合的肾脏样本作为单个生物学重复。从上述混合样本中分离总RNA，经一轮扩增后用于Affymetrix基因芯片检测。