Isolation of new genes related to hematopoietic cells using gene trap screening in ES cells

我们构建了一个基因陷阱载体，该载体包含En-2剪接受体位点、IRES信号、LacZ基因作为报告基因和肌动蛋白启动子控制下的新霉素抗性基因。该载体通过使用视黄酸作为体外ES细胞分化的因子而有效地工作。我们将该载体导入D3或E14-1 ES细胞，并建立了数千个克隆。我们将这些克隆培养在来自op/op小鼠的OP-9饲养层上，并在培养的第8天用X-gal染色。虽然检测到很少的LacZ阳性克隆，我们挑选这些细胞，并从这些阳性克隆的cDNA文库。我们对10多个cDNA克隆进行了测序，并鉴定了一些未知的基因片段。北方印迹分析表明，大部分基因在成年小鼠组织中广泛表达，但也有少数基因具有一定的组织特异性表达。ES细胞在体外可分化为不同的组织，但这些细胞是异质性的。因此，我们试图摸索出支持胚胎干细胞体外分化为特定谱系的培养条件，或利用谱系特异性表面标记通过流式细胞仪分选出分化的细胞，为将来建立其他物种的胚胎干细胞基因捕获技术奠定基础。

We constructed a gene trap vector contained a En-2 splice acceptor site, IRES signal, the LacZ gene as a reporter gene and neomycin resistant gene under the control of an actin promoter. This vector works effectively by using retinoic acid as a factor for ES cell differentiation in vitro . We introduced this vector into D3 or E14-1 ES cells and established several thousands of clones. We cultured these clones on OP-9 feeder layers derived from op/op mice and stained with X-gal at day 8 of culture. Although very few LacZ positive clones were detected, we picked up these cells and made cDNA library from these positive clones. We sequenced more than 10 cDNA clones and identified some unknown gene fragment. Most of them were ubiquitously expressed in adult mouse tissues revealed by Northern blot analysis but a few showed tissue specific expression to some extent. But none of these clones were expressed exclusively in hematopoietic tissues.ES cells differentiate into different tissues in vitro, but these are heterogeneous. Therefore, we are trying to fish out culture conditions which support ES cell differentiate to specific lineage in vitro or sorting out differentiated cells using lineage specific surface markers by FACS.ES cells established from other species can be used in this gene trap approach in the future.