Gene expression changes in embryonic PECAM-1+ cells from Gata2 +9.5 enhancer targeted mice

We have generated a nonconditional deletion of an E-box - GATA motif composite element from a putative enhancer 9.5 kb downstream of the Gata2 1S promoter. The mutation results in lethality at E13.5 to E14.5 with embryos exhibiting dectects in definitive hematopoiesis and vasculature integrity. Hematopoietic stem and progenitor cells are nearly absent from these mice and Gata2 expression is severely reduced in Pecam-1+ endothelial cells from E12.5-12.5 embryos but is expressed at normal levels in the embryonic brain. We have employed gene expression profiling in Pecam-1+ cells from wild type and Gata 2+9.5 mutant littermates to identify genes involved in maintaining vascular integrity. The Gata2+9.5 mutation was backcrossed into the C57Bl/6J background. Anti-CD31 antibody bound to magnetic beads was used to enrich for PECAM-1+ cells from four Gata2+9.5 E12.5 embryos and four wild type littermates from two litters. A single RNA sample from each embryo was used for array hybridization.我们针对Gata2基因1S启动子下游9.5kb处的推定增强子区域，构建了E框（E-box）-GATA基序（GATA motif）复合元件的非条件性缺失突变。该突变可导致胚胎在胚胎发育第13.5天至第14.5天（E13.5~E14.5）阶段死亡，突变胚胎表现出定型造血（definitive hematopoiesis）与血管完整性缺陷。此类突变小鼠体内几乎无法检测到造血干细胞和祖细胞（hematopoietic stem and progenitor cells），且在胚胎发育第12.5天的Pecam-1（血小板内皮细胞黏附分子1，PECAM-1）阳性内皮细胞中，Gata2的表达量显著降低，但在胚胎脑组织中其表达水平仍维持正常。为鉴定参与维持血管完整性的基因，我们对野生型与Gata2+9.5突变型同窝仔鼠的Pecam-1阳性细胞开展了基因表达谱分析。本研究中将Gata2+9.5突变回交至C57BL/6J遗传背景。实验采用磁珠偶联抗CD31抗体，从两窝共4只Gata2+9.5突变型E12.5胚胎及4只同窝野生型胚胎中富集PECAM-1阳性细胞；每个胚胎的单个RNA样本均用于芯片杂交（array hybridization）实验。