Molecular mechanisms for hemato-vascular formation

我们建立了绿色荧光蛋白(GFP)基因敲除小鼠(G2-GFP)，将GFP基因插入到Gata2基因的造血特异性启动子/第一外显子下，并对表达GFP的细胞进行了表型分析。骨髓单个核细胞中GFP阳性细胞占0.5%。这些GFP阳性细胞表达c-Kit，没有任何染色谱系标记(LIN)，在OP9细胞层有可能形成多个谱系，提示GFP阳性细胞既属于造血祖细胞群，也属于造血干细胞群。细胞增殖相关分子c-Myc、Cyclin A2和Cyclin B1在GFP阳性细胞中的表达高于GFP阴性细胞。此外，LIN-/c-Kit^+/GFP^+细胞中有30%以上的细胞聚集在S/G2/M期。这些发现表明GATA-2在反式…中起着重要作用为了进一步研究GATA-2在体内对造血细胞增殖的作用，我们建立了GATA-2基因条件敲除等位基因(G2-CKO)，将GATA-2基因插入到GATA-2基因翻译起始点的两个loxP序列之间。由于GATA-2基因敲除小鼠具有胚胎致死性，因此该G2-CKO小鼠品系可能是检测GATA-2在骨髓造血中作用的最合适的手段之一。用干扰素诱导Cre重组酶表达的Mx1-Cre转基因小鼠与GATA-2靶向小鼠(G2^&lt；CKO/GFP&gt；：：Mx1-Cre)杂交，成功构建了三重复合小鼠克隆(Gata2^&lt；CKO/GFP&gt；：：Mx1-Cre)。我们现在已经准备好进行骨髓移植分析，使用这些小鼠的骨髓细胞来检查GATA-2在成人造血中的功能。较少

We have established green fluorescent protein (GFP) knock-in knockout mice (G2-GFP), in which the GFP gene was inserted into Gata2 gene under the hematopoietic specific promoter/ first exon and analyzed the phenotypes of GFP expressing cells. The percentage of GFP positive cells was 0.5 % in the bone marrow mononuclear cells. These GFP positive cells, expressing c-Kit without any staining lineage markers (Lin), had a potential to develop several lineage committing cells on the OP9 cell layer, suggesting that GFP-positive cells were belonging to either the population of hematopoietic progenitor cells or that of hematopoietic stem cells. The expression of cellular proliferation-related molecules, such as c-Myc, Cyclin A2 and Cyclin B1, in the GFP-positive cells were higher than those in the GFP-negative cells. In addition, more than 30 % of the cells in the population of Lin-/c-Kit^+/GFP^+ accumulated in S/G2/M phase. These findings indicated that GATA-2 had an important role for the tra … More nscriptional regulation of genes relating to cell cycle progression of the immature hematopoietic progenitor cells.To further investigate the function of GATA-2 on the hematopoietic cell proliferation in vivo, we established Gata2 gene conditional knockout allele (G2-CKO), in which GATA-2 cDNA sandwiched between two loxP sequences was inserted into the translation initiation site of Gata2 gene. Because the simple Gata2 gene knockout mice were embryonic lethal, this G2-CKO mouse line might be one of the most appropriate means to examine the function of GATA-2 on bone marrow hematopoiesis. We intercrossed the compound GATA-2 targeting mice (G2^<CKO/GFP>) with Mx1-Cre transgenic mice, in which the expression of Cre recombinase was induced by the treatment of interferon, and successfully generated the triple compound mouse colony (Gata2^<CKO/GFP>::Mx1-Cre). We are now ready for bone marrow transplantation analyses using the bone marrow cells of these mice to examine the function of GATA-2 for adult hematopoiesis. Less