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

我们构建了一个包含En-2剪接受体位点、IRES信号、LacZ基因作为报告基因和在肌动蛋白启动子控制下的新霉素耐药基因的基因诱捕载体。该载体利用视黄酸作为胚胎干细胞体外分化因子，能有效地发挥作用。我们将该载体导入D3或E14-1 ES细胞，建立了数千个克隆。我们将这些克隆培养在op/op小鼠的op -9饲养层上，并在培养第8天用X-gal染色。虽然检测到的LacZ阳性克隆非常少，但我们挑选了这些细胞并从这些阳性克隆中构建了cDNA文库。我们对10多个cDNA克隆进行了测序，并鉴定出一些未知的基因片段。Northern blot分析显示，这些基因在成年小鼠组织中普遍表达，但也有少数基因有一定的组织特异性表达。但这些克隆没有一个在造血组织中完全表达。胚胎干细胞在体外分化成不同的组织，但这些组织是异质的。因此，我们正在尝试在体外寻找支持胚胎干细胞向特定谱系分化的培养条件，或通过FACS利用谱系特异性表面标记对分化的细胞进行分类。从其他物种建立的胚胎干细胞将来可以用于这种基因陷阱方法。

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.