Expression data from undifferentiated and differentiated mouse female ES cells PGK12.1

Affymetrix 430 2.0 mouse arrays were used for expression analyses in undifferentiated and differentiated PGK12.1 ES cells. We found that the X:autosome expression ratios calculated from the mean expression values of X-linked and autosomal genes from microarrays was ~1.4 in undifferentiated female ES cells and then decreased to 1.2 in PGK12.1 cells after 15-day embryoid body differentiation. Thus, a substantial level of X upregulation is already evident in these ES cells prior to differentiation. Our findings based on Affymetrix expression arrays are consistent with microarray analysis from other labs and our RNA-seq analysis of mouse female PGK12.1 ES cells. Mouse female ES cells PGK12.1 were differentiated by the EB (embryoid body) differentiation protocol. The presence of two active X chromosomes in undifferentiated female ES cells and one active X in 15-day differentiated cells was verified by Xist RNA FISH. Total RNA was prepared from undifferentiated and 15-day differentiated PGK12.1 and was used for expression array analyses to examine exprssion of the X chromosome during differentiation.

本研究采用Affymetrix 430 2.0小鼠基因芯片（Affymetrix 430 2.0 mouse arrays），对未分化及分化后的PGK12.1胚胎干细胞（Embryonic Stem cells, ES）进行表达谱分析。研究人员通过芯片中X连锁基因与常染色体基因的平均表达值，计算得到X染色体与常染色体的表达比值：未分化的雌性ES细胞中该比值约为1.4；经15天胚胎体（Embryoid Body, EB）分化的PGK12.1细胞中，该比值降至1.2。由此表明，在分化前的ES细胞中，已存在显著的X染色体上调水平。本研究基于Affymetrix表达芯片得到的结果，与其他实验室的芯片分析结果及本团队对雌性小鼠PGK12.1 ES细胞的RNA测序（RNA-seq）分析结果相符。本研究采用EB分化方案诱导雌性小鼠PGK12.1 ES细胞分化，并通过Xist RNA荧光原位杂交（RNA Fluorescence In Situ Hybridization, RNA FISH）验证：未分化的雌性ES细胞中存在两条活性X染色体，而经15天分化后的细胞仅保留一条活性X染色体。研究人员从未分化及经15天分化的PGK12.1细胞中提取总RNA，用于表达芯片分析，以探究细胞分化过程中X染色体的表达变化。