Single cell microarray analysis of mouse primordial germ cells with Prdm14 mutation.

Prdm14 is a critical gene for specifying mouse primordial germ cells (PGCs). The changes in expression in mouse PGCs caused by mutations of the Prdm14 gene were investigated at the single-cell level using microarray analysis. C57BL/6 mice with the Prdm14 (+/-) genotype (Yamaji et al., Nature Genetics, 2008) were crossed. Pregnant mice were sacrificed at embryonic day (E) 7.25 - 7.5 and embryos at the early-to-mid allantois bud stage were isolated. The base of the allantois bud, where primordial germ cells (PGCs) were clustered, was dissected using a glass knife and cells were dissociated with trypsin as described (Kurimoto et al., Genes and Development, 2008). Single cells were randomly isolated manually using a glass capillary as described (Kurimoto et al., Nature Protocols, 2007) and directly subjected to cDNA amplification (Kurimoto et al., Nucleic Acids Research, 2006). cDNAs of PGCs were identified based on Prdm1 expression using targeted PCR as described (Kurimoto et al., Genes and Development, 2008). Genotypes of Prdm14 were identified by PCR as described (Yamaji et al., Nature Genetics, 2008). cDNAs of PGCs with Prdm14 (+/+), (+/-), and (-/-) genotypes were then subjected to microarray analysis using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. Microarray data was processed using dChip (Ver.1.3) with the PM/MM difference model. Expression level of Dppa3 (1424295_at) were investigated.

Prdm14是特化小鼠原始生殖细胞（primordial germ cells, PGCs）的关键基因。本研究采用单细胞微阵列分析技术，探究了Prdm14基因突变对小鼠原始生殖细胞基因表达谱的影响。
携带Prdm14 (+/-)基因型的C57BL/6小鼠（Yamaji等，《自然·遗传学》，2008）经杂交繁育。于胚胎发育第7.25~7.5天处死孕鼠，分离早中期尿囊芽阶段的胚胎。参照Kurimoto等2008年发表于《基因与发育》的方法，使用玻璃刀切割聚集有原始生殖细胞的尿囊芽基部，再通过胰蛋白酶消化解离细胞。随后采用玻璃毛细管手动随机分离单个细胞（Kurimoto等，《自然·实验方案》，2007），并直接进行cDNA扩增（Kurimoto等，《核酸研究》，2006）。
参照Kurimoto等2008年《基因与发育》的方法，基于Prdm1的表达特征鉴定原始生殖细胞的cDNA；同时通过PCR技术鉴定样本的Prdm14基因型（Yamaji等，《自然·遗传学》，2008）。
随后对携带Prdm14 (+/+)、(+/-)及(-/-)基因型的原始生殖细胞cDNA，采用Affymetrix GeneChip小鼠基因组430 2.0芯片开展微阵列分析。微阵列数据采用dChip（版本1.3）结合PM/MM差异模型进行处理。本研究同时检测了探针编号为1424295_at的Dppa3基因的表达水平。