Comparison of mRNA expression between wildtype and Wnt9b-/- isolated metanphric mesenchyme from E11.5 kidneys.

Wnt9b is expressed in the ureteric bud of the kidney at all stages of development. In Wnt9b mutants, the ureteric bud forms but the metanephric mesenchyme is never induced to undergo differentiation. We used microarrays to compare differences in gene expression between wildtype and Wnt9b mutants. To focus on direct targets, mesenchymes isolated away from the E11.5 ureteric bud were assayed Ell.5 kidneys were manually dissected away from embryos collected from Wnt9b+/- intercrosses. Mesenchymes were isolated away from the ureteric bud after a brief enzymatic digestion (trypsin/pancreatin). Paired mesenchymes were stored int RNA-Later until the genotype was determined. 25-30 Wnt9b-/- or Wnt9b+/- mesenchymes were pooled for each replicate (giving approx. 1ug of total RNA). cRNA was synthesized and hybridized to the Affymetrix GeneChip Mouse Genome 430.2 Array.Wnt9b在肾脏发育的所有阶段均表达于输尿管芽（ureteric bud）中。在Wnt9b突变体中，输尿管芽虽可形成，但后肾间充质（metanephric mesenchyme）始终无法被诱导分化。本研究采用基因表达微阵列（microarrays）技术，比较野生型与Wnt9b突变体的基因表达差异。为聚焦直接靶基因，研究人员针对脱离E11.5（胚胎发育第11.5天）期输尿管芽的间充质开展检测：从Wnt9b+/-互交繁育的胚胎中手动分离E11.5期肾脏；经短暂的胰蛋白酶/胰酶素（trypsin/pancreatin）酶消化后，将间充质与输尿管芽分离；将配对的间充质样本保存于RNA-Later试剂中，直至完成基因型鉴定。每个生物学重复样本混合25~30个Wnt9b-/-或Wnt9b+/-的间充质样本，最终获得约1 μg总RNA。随后合成互补RNA（cRNA），并将其与Affymetrix GeneChip小鼠基因组430.2芯片进行杂交。