Hassan M, Mirmohammadsadegh A, Selimovic D, Nambiar S, Tannapfel A, Hengge U R
Institute of Pathology, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.
Cell Mol Life Sci. 2005 Sep;62(17):2015-26. doi: 10.1007/s00018-005-5172-6.
We describe a general strategy for the identification of functional genes that, when downregulated, result in a selectable phenotype. This strategy is based on expression selection of cDNA fragments that counteract their cognate genes. A cDNA library containing random fragments expressed in human HepG2, A375 and CLS-354 cells was used to identify functional genes whose inhibition conferred resistance to Fas-induced apoptosis. Thirty-five clones were isolated, 28 of which were derived from unknown genes, that tagged 19 individual genes and 7 of which referred to known genes that tagged the apoptosis-related protein (APR)-1, -2 and indoleamine-pyrrole 2,3,-dioxygenase (IDO). The ability of APR-1-, -2- and IDO-derived antisense RNAs to induce resistance to Fas in HepG2, A375 and CLS-354 cells suggested that APR-1, -2 and IDO genes are involved in the machinery of Fas-mediated apoptosis. Our gene discovery strategy provides a generally applicable procedure to identify functional genes that interfere with apoptosis, and may therefore be clinically relevant for tumor therapy.
我们描述了一种识别功能基因的通用策略，这些基因下调时会导致可选择的表型。该策略基于对与同源基因相互作用的cDNA片段进行表达筛选。利用一个在人肝癌细胞系HepG2、黑色素瘤细胞系A375和结肠癌细胞系CLS-354中表达随机片段的cDNA文库，来识别那些抑制后能赋予对Fas诱导凋亡抗性的功能基因。分离出35个克隆，其中28个来自未知基因，标记了19个独立基因，7个来自已知基因，标记了凋亡相关蛋白（APR）-1、-2和吲哚胺-吡咯2,3-双加氧酶（IDO）。源自APR-1、-2和IDO的反义RNA在HepG2、A375和CLS-354细胞中诱导对Fas抗性的能力表明，APR-1、-2和IDO基因参与了Fas介导的凋亡机制。我们的基因发现策略提供了一个普遍适用的程序来识别干扰凋亡的功能基因，因此可能在肿瘤治疗中具有临床相关性。
