Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293 T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.

中文翻译：

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基于piggyBac的双诱导二元载体系统：研究基因功能和相互作用的新型通用平台。

真核生物诱导型过表达系统，包括Tet-On和米非司酮诱导型系统，已被广泛用于通过反向遗传学研究基因功能。在迄今为止报道的转座子系统中，piggyBac转座子系统是培养的哺乳动物细胞中最有效的系统之一。在这里，我们报告了一个基于piggyBac的双重诱导系统，该系统结合了先前系统的优点。为了创建该系统，将Tet-On和米非司酮诱导型系统的反式和顺式元件分别克隆到基于piggyBac的反式载体和顺式载体中。将RUNX1的两个剪接变体RUNX1a和RUNX1b的编码区插入顺式载体，以测试其与荧光标记蛋白一起表达外源基因的能力。建立了转基因293 T细胞，并且通过用DOX和/或米非司酮诱导外源基因的表达来测试该系统；GFP和/或mCherry用作报告基因。通过qRT-PCR和Western blot检测，该系统有效，严格地诱导了GFP / mCherry及其共表达基因的表达，而没有明显的相互干扰。这个基于piggyBac的双重诱导系统代表了一种新的遗传工具，可用于研究几乎所有生物体内和体外的基因功能和相互作用。