Romprey Matt J, Armstrong Raveen, McGill Peter, Klingbeil Michele M
Department of Microbiology, University of Massachusetts, Amherst, MA United States of America.
Institute for Applied Life Sciences, University of Massachusetts, Amherst, MA United States of America.
bioRxiv. 2025 Sep 2:2025.08.31.673413. doi: 10.1101/2025.08.31.673413.
is a model unicellular parasite for cellular and molecular genetic studies, but tools for more multiplexed experiments are limited. Tetracycline (Tet)-inducible gene regulation has been a long-standing and effective approach for overexpression, RNAi and genome wide screens. The single inducer Tet-On system has been foundational for studying essential genes that are required for biological processes and identifying potential drug targets. To achieve greater flexibility in experimental design, we capitalized upon previously described dual inducer systems that combined vanillic acid (Van) or cumate (Cym) with Tet as inducers. Here we report the development of a triple inducer system combining Cym, Van and Tet repressor elements to selectively regulate the expression of three genetic elements within a single cell line called PHITER. As proof of principle to demonstrate independent control, we adapted the previously characterized Van/Tet dual inducer RNAi complementation cell line, IBComp for additional Cym inducible expression of an eGFP reporter. We provide evidence that each of the inducer elements operate independently with no evidence of leaky expression. In this system, Cym induction is specific and tunable. Additionally, simultaneous triple induction for RNAi complementation with wildtype POLIB and eGFP expression phenocopied the near complete rescue of RNAi previously reported. This system enables the study of complex and pleiotropic phenotypes through more sophisticated experimental design.
是细胞和分子遗传学研究的典型单细胞寄生虫，但用于更多多重实验的工具有限。四环素（Tet）诱导的基因调控一直是用于过表达、RNA干扰和全基因组筛选的长期有效方法。单一诱导剂Tet-On系统一直是研究生物过程所需必需基因和鉴定潜在药物靶点的基础。为了在实验设计中实现更大的灵活性，我们利用了先前描述的将香草酸（Van）或香豆酸（Cym）与Tet作为诱导剂相结合的双诱导剂系统。在此，我们报告了一种三诱导剂系统的开发，该系统结合了Cym、Van和Tet阻遏元件，以在称为PHITER的单一细胞系中选择性调节三个遗传元件的表达。作为证明独立控制的原理验证，我们对先前表征的Van/Tet双诱导剂RNA干扰互补细胞系IBComp进行了改造，用于额外的Cym诱导的eGFP报告基因表达。我们提供的证据表明，每个诱导元件都独立运行，没有渗漏表达的迹象。在这个系统中，Cym诱导是特异性的且可调节的。此外，同时进行三诱导以实现与野生型POLIB的RNA干扰互补和eGFP表达，模拟了先前报道的RNA干扰几乎完全挽救的情况。该系统能够通过更复杂的实验设计来研究复杂和多效性表型。
