Gene-trap expression screening to identify endothelial-specific genes
Gene-trap expression screening to identify endothelial-specific genes
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DOI：
10.1182/blood-2004-01-0254
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发表时间：
2004-08-01
期刊：
影响因子：
20.3
通讯作者：
Rossant, J
中科院分区：
文献类型：
--
作者：
Hirashima, M;Bernstein, A;Rossant, J
内皮细胞是血管形成的关键细胞成分。内皮细胞中表达的许多信号受体在胚胎发生过程中的血管发育中起着关键作用。然而，血管分化所需的下游反应基因仍然没有明确确定。在这里，我们描述了一个协议的发展基因陷阱表达筛选胚胎干细胞（ES）内皮细胞特异性基因。ES细胞在96孔板中的OP9饲养细胞层上分化为内皮细胞。在初步筛选中，在筛选的864个ES克隆中，5个基因捕获的ES细胞系显示出基因捕获lacZ报道基因的上调表达。其中一个被捕获的基因是内皮糖蛋白，一种内皮特异性转化生长因子β III型受体，另一个是ASPP 1，一种p53结合蛋白。体内表达分析的lacZ报告证实，这两个基因特异性表达在内皮细胞在早期小鼠胚胎发生。因此，基因捕获表达筛选可用于鉴定早期内皮特异性基因并分析其在小鼠中的功能。
The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. in a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-beta type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.