Transcription profiling of mouse protocadherin 12 knockout

Protocadherin 12 (Pcdh12) is a transmembrane adhesive protein with homophilic adhesive properties and expressed in endothelial cells, the glycogen trophoblast cells of the placenta, and the mesangial cells of kidney glomeruli. Pcdh12-deficient mice are alive although they show alterations in placenta development. To reveal Pcdh12-associated pathways, 45,000 - Affymetrix probe arrays were used on Pcdh12 knockout and control placentas. Experiment Overall Design: To identify genes whose expression was modified by Pcdh12 deficiency, we examined global gene expression profiles of whole E12.5 knockout placentas (Pcdh12 -l-) and their wild-type (WT - Pcdh12+l+ ) littermates, using the Affymetrix Murine Genome 430 2.0 microarrays. Gestation day 12 was chosen as it is the period at which placentas already formed distinct layers, but organogenesis is still very active. Total RNA was extracted from five Pcdh12 knockout embryos and five normal placentas. RNA were tested for integrity on Agilent 2100 BioAnalyzer, prior to cRNA synthesis and chip hybridization (performed by the Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France).原钙粘蛋白12（Protocadherin 12，Pcdh12）是一种具备同嗜性黏附特性的跨膜黏附蛋白，在内皮细胞、胎盘糖原滋养层细胞以及肾小球系膜细胞中均有表达。Pcdh12基因敲除小鼠仍可存活，但会出现胎盘发育异常。 为揭示Pcdh12相关的调控通路，研究人员对Pcdh12敲除胎盘与对照胎盘使用了Affymetrix 45000探针阵列。 实验总体设计：为鉴定受Pcdh12缺失调控的差异表达基因，本研究采用Affymetrix小鼠基因组430 2.0微阵列（Affymetrix Murine Genome 430 2.0 microarrays），对全胚胎12.5天（E12.5）的Pcdh12纯合敲除胎盘（Pcdh12⁻/⁻）及其同窝野生型（WT，Pcdh12⁺/⁺）幼崽的全基因表达谱进行分析。选择妊娠第12天作为实验节点，是因为此时胎盘已形成清晰的结构分层，但器官发生过程仍处于高度活跃的阶段。 研究人员从5个Pcdh12敲除胚胎与5个正常胎盘中提取总RNA。在进行互补RNA（cRNA）合成与芯片杂交前，使用Agilent 2100生物分析仪（Agilent 2100 BioAnalyzer）对RNA的完整性进行检测。本实验由法国斯特拉斯堡遗传学与分子细胞生物学研究所完成。