Differential pre-mRNA processing in Crebbp+/- mice. Mus musculus

The presence of unspliced transcripts in hematopoietic stem cells (HSCs) and the proposed association of CREBBP with the constitutive production of unspliced RNA and with pre-mRNA processing prompted us to examine more closely an anomaly we had noted in microarray-based gene expression studies but had previously attributed to experimental noise. We noticed that more than half of the probe sets down-regulated in Crebbp+/- fetal liver HSCs (FLHSCs) relative to wild-type (WT) mapped entirely within introns, rather than detecting exonic or spliced sequences. We therefore set out to test whether this might be evidence that reduced CREBBP levels selectively alter the generation of full-length, unspliced pre-mRNA. We also asked whether this process might be associated with differentiation since self-renewal and lineage commitment are the both responses for which HSCs are primed. Overall design: Total RNA from wild-type, Crebbp+/-, Ep300+/-, Cdkn1a-/- FLHSCs and from wild type and Crebbp+/- mouse embryonic fibroblasts (MEFs) was isolated and hybridized to Affymetrix Mouse 430 2.0 expression microarrays. Fetal liver HSC RNA was amplified using the Ovation kit prior to hybridization. cell type comparison

造血干细胞（hematopoietic stem cells, HSCs）中存在未剪接转录本，且已有研究提出CREBBP与未剪接RNA的组成型生成及前体mRNA（pre-mRNA）加工存在关联，这促使我们对此前在基于微阵列的基因表达研究中观测到、但曾被归因于实验噪声的异常现象展开更细致的探究。我们注意到，相较于野生型（wild-type, WT）样本，Crebbp+/-胎肝造血干细胞（fetal liver HSCs, FLHSCs）中超过半数的下调探针组完全定位于内含子区域，而非靶向外显子或剪接序列。因此，我们着手验证这一现象是否能够证明CREBBP水平降低会选择性改变全长未剪接前体mRNA的生成过程。鉴于自我更新与谱系定向分化是造血干细胞预编程的两种核心应答方向，我们同时探究该异常过程是否与细胞分化相关。实验整体设计：提取野生型、Crebbp+/-、Ep300+/-、Cdkn1a-/-胎肝造血干细胞，以及野生型与Crebbp+/-小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）的总RNA，将其与Affymetrix小鼠430 2.0表达谱微阵列芯片进行杂交。胎肝造血干细胞的总RNA在杂交前采用Ovation试剂盒进行扩增。细胞类型比较