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首页> 外文期刊>Cell cycle >Single-vector inducible lentiviral RNAi system for oncology target validation.
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Single-vector inducible lentiviral RNAi system for oncology target validation.

机译：单载体诱导型慢病毒RNAi系统，用于肿瘤学目标验证。

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摘要

The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.
机译：RNA干扰（RNAi）的使用已使哺乳动物癌细胞的功能丧失研究成为可能，因此对于鉴定和验证癌症药物靶标至关重要。但是，当前的瞬时siRNA和稳定的shRNA系统分别由于其短暂的作用和有限的体内效用而在准确评估癌症依赖性方面的应用有限。在这项研究中，生成了单载体慢病毒，Tet诱导型shRNA系统（pLKO-Tet-On），可以快速生成具有可调节shRNA表达的多种稳定细胞系。我们通过针对两种癌细胞系中的两个多梳子组蛋白Bmi-1和Mel-18展示了该系统的优势和多功能性。我们的数据表明，pLKO-Tet-On介导的敲低受诱导剂四环素及其衍生物强力霉素的浓度和时间依赖。此外，在诱导剂退出后靶基因表达被完全恢复。在所有情况下，诱导性shRNA还靶向了另外17种不同的基因产物。重要的是，我们在功能上验证了pLKO-Tet-On载体在体内可逆沉默靶转录本的能力。因此，本文报道的通用且耐用的诱导型慢病毒RNAi系统可以用作快速揭示肿瘤细胞依赖性的强大工具。

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