Gene expression differences between Gata2cko and wt mouse rhombomere 1

During development, rhombomere 1 gives rise to a multitude of cell types, including serotonergic as well as glutamatergic neurons that populate the raphe nuclei. Transcription factor Gata2 has been shown to regulate the development of the serotonergic neurons in the rhombomere 1. To further understand the role and targets of Gata2 in the rhombomere 1 during development, we used cDNA microarrays to compare gene expression in the embryonic day 12.5 (E12.5) rhombomere 1 tissues extracted from wild-type and Gata2 mutant embryos. Rhombomere 1 region was dissected from the E12.5 mouse embryos from the En1-cre, Gata2 flox/+ x Gata2 flox/flox crosses, and frozen in liquid nitrogen. After genotyping, dissected rhombomere 1 pieces were pooled according to genotype, combining 6 En1-cre, Gata2 flox/+ samples into one control pool, and 6 En1-cre, Gata2 flox/flox samples as one mutant pool. RNA was extracted from 3 sample pools (biological replicates) per genotype, reverse transcribed and labelled according to standard Illumina protocols and hybridized on Illumina Mouse WG-6 Expression BeadChip arrays. For the comparison of gene expression, samples were grouped according to genotype (control vs mutant).在发育进程中，菱脑节1（rhombomere 1）可分化产生多种细胞类型，包括定位于中缝核（raphe nuclei）的血清素能神经元（serotonergic neurons）与谷氨酸能神经元（glutamatergic neurons）。已有研究证实，转录因子Gata2（transcription factor Gata2）可调控菱脑节1内血清素能神经元的发育过程。为进一步阐明Gata2在发育阶段于菱脑节1中的作用及靶基因，我们采用cDNA微阵列（cDNA microarrays）技术，对比分析野生型与Gata2突变体胚胎的胚胎第12.5天（embryonic day 12.5，E12.5）菱脑节1组织的基因表达谱。实验样本取自En1-cre、Gata2 flox/+ × Gata2 flox/flox交配获得的E12.5小鼠胚胎，分离菱脑节1区域后置于液氮中速冻保存。完成基因分型后，根据基因型对分离得到的菱脑节1组织块进行混样：将6份En1-cre、Gata2 flox/+样本整合为1个对照混样，6份En1-cre、Gata2 flox/flox样本整合为1个突变体混样。每个基因型设置3个样本混样作为生物学重复，从中提取RNA，按照标准Illumina实验流程进行反转录与标记，随后在Illumina Mouse WG-6 Expression BeadChip arrays上完成杂交。本研究的基因表达对比分析将按照基因型划分为对照组与突变组进行。