Transcription profiling by array of wild type and HoxA11 metenephric mesenchyme and ureteric bud from embryonic day 11.5 mice

E11.5 metanephric mesenchyme and ureteric bud were dissected from the E11.5 kidney rudiment using fine manual microdissection (ureteric bud only) or both fine manual microdissection and laser capture microdissection (metanephric mesenchyme) to define the gene expression profiles of these structures. Additionally, HoxA11, HoxD11 compound null E11.5 metanephric mesenchyme was obtained through laser capture microdissection allowing analysis of possible Hox targets in kidney development. Targets from multiple biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430_v2 arrays. Using microdissection techniques, ureteric bud and metanephric mesenchyme were dissected from E11.5 kidney rudiments allowing the identificated genes specifically regulated in either structure. In addition, Hoxa11, Hoxd11 compound null E11.5 metanephric mesenchyme were normalized to wild type embryonic controls allowing the identification of potential Hox targets in normal kidney development. Each structure/genotype were represented in biological (separate embryo) replicate.本研究通过精细手工显微切割（fine manual microdissection，仅用于分离输尿管芽（ureteric bud）），或联合精细手工显微切割与激光捕获显微切割（laser capture microdissection）以获取后肾间充质（metanephric mesenchyme），从E11.5期肾原基（kidney rudiment）中分离得到E11.5期后肾间充质与输尿管芽，以明确这两种结构的基因表达谱。此外，通过激光捕获显微切割获取HoxA11、HoxD11复合纯合缺失的E11.5期后肾间充质，用于分析肾脏发育过程中潜在的Hox靶基因。针对每组样本的多个生物学重复（biological replicate）生成靶基因相关数据，并采用Affymetrix MOE430_v2基因芯片检测基因表达谱。通过显微切割技术从E11.5期肾原基中分离输尿管芽与后肾间充质，以此鉴定在两种结构中特异性调控的基因；此外，将Hoxa11、Hoxd11复合纯合缺失的E11.5期后肾间充质的表达数据与野生型胚胎对照进行标准化处理，以鉴定正常肾脏发育过程中潜在的Hox靶基因。每一种结构/基因型均设置生物学重复，即采用来自不同胚胎的样本。