Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
PLoS One. 2009 Sep 15;4(9):e7029. doi: 10.1371/journal.pone.0007029.
The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.
We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.
Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.
宿主防御入侵病原体所需的事件涉及由许多衔接分子、激酶和转录因子组成的复杂信号级联，最终导致促炎细胞因子（如肿瘤坏死因子-α（TNF-α））的产生。这些信号级联如何被调节，以及参与的蛋白质和调节因子仍知之甚少。
我们在此报告了一种完全随机短发夹 RNA（shRNA）文库的开发，以及一种新的正向遗传筛选策略，以鉴定 Toll 样受体（TLR）依赖性促炎反应的抑制剂。我们开发了一种稳定转染表达白喉毒素-A（DT-A）的构建体的小鼠巨噬细胞报告细胞系，该构建体受 TNF-α 启动子的控制。用 TLR 配体脂多糖（LPS）刺激报告细胞系会导致 DT-A 诱导的细胞死亡，而添加针对 TLR 衔接分子 MyD88 的 shRNA 可预防这种细胞死亡。利用这种细胞系，我们筛选了完全随机的慢病毒短发夹 RNA（shRNA）文库，以寻找抑制 TLR 介导的 TNF-α 产生的序列。从存活细胞中回收 shRNA 序列可鉴定出显著抑制 TLR4 依赖性 TNF-α 基因表达的独特 shRNA 序列。此外，这些 shRNA 序列特异性阻断 TLR2 但不阻断 TLR3 依赖性 TNF-α 的产生。
因此，我们描述了生成新工具的方法，以促进哺乳动物细胞中的大规模正向遗传筛选，并鉴定出 TLR2 和 TLR4 依赖性促炎反应的有效 shRNA 抑制剂。
