Clinic of Dermatology, University Hospital of Düsseldorf, Germany.
Eur J Med Res. 2010 Apr 8;15(4):162-8. doi: 10.1186/2047-783x-15-4-162.
The sequences of many genomes are available; therefore, relevant methods are needed for rapid and efficient identification of functional genes. The ability of tumour cells to resist apoptosis induced by anticancer agents may decide about the success or failure of tumour elimination. Although the CD95-signaling pathway is functional in tumour cells, the increased resistance of tumour cells to CD95-mediated apoptosis has been widely reported. In order to identify genes that might determine the response of tumour cells to CD95-mediated apoptosis, we modified the conventional technical knock out (TKO) strategy for isolation of genes that function in CD95-mediated apoptosis. Due to the fact that multiple different plasmids are usually introduced into the same cells, the effectiveness of the conventional TKO strategies is low. To overcome this obstacle, we replaced the conventional TKO strategy (based on stably expressed randomly fragmented cDNA libraries) with a multi-cycle selection procedure (based on transiently expressed randomly fragmented cDNA libraries with multi-cycle selection). Using this approach we could rapidly and significantly identify small numbers of antisense mRNA molecules, whose re-introduction into different tumour types confirmed their ability to block the pro-apoptotic function of their cognate genes. Thus, our modified TKO strategy provides a generally applicable procedure for the identification of functional genes with pro-apoptotic properties that may be clinically relevant to tumour therapy.
许多基因组的序列已经可用；因此，需要快速有效的方法来识别功能基因。肿瘤细胞抵抗抗癌药物诱导的细胞凋亡的能力可能决定肿瘤消除的成败。尽管肿瘤细胞中 CD95 信号通路是功能性的，但肿瘤细胞对 CD95 介导的细胞凋亡的抵抗力增加已被广泛报道。为了鉴定可能决定肿瘤细胞对 CD95 介导的细胞凋亡反应的基因，我们修改了传统的技术敲除（TKO）策略，用于分离在 CD95 介导的细胞凋亡中起作用的基因。由于通常将多个不同的质粒引入同一细胞中，因此传统的 TKO 策略的效率较低。为了克服这一障碍，我们用多循环选择程序（基于瞬时表达的随机片段化 cDNA 文库，带有多循环选择）代替了传统的 TKO 策略（基于稳定表达的随机片段化 cDNA 文库）。使用这种方法，我们可以快速显著地鉴定出少量的反义 mRNA 分子，将它们重新引入不同的肿瘤类型证实了它们阻断其同源基因促凋亡功能的能力。因此，我们修改后的 TKO 策略为鉴定具有促凋亡特性的功能基因提供了一种普遍适用的方法，这些基因可能与肿瘤治疗的临床相关。
