Abstract
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.
Highlights
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis
We fused a hormone-binding domain of insect ecdysone receptor (EcR) to the full length QF transcriptional activator (TA) at its C-terminus to gain nuclear access only when the chimera binds to the ecdysone agonist, tebufenozide (Teb, known as RH-5992), a lipophilic molecule that is non-toxic to developing zebrafish (Fig. 1a, b)[3,23,24]
Though the middle domain truncated form of QF was reported to be nontoxic in Drosophila[26], it was still detrimental to zebrafish embryos and unacceptably leaky (Fig. 1a–c, Supplementary Fig. 1 and Supplementary Table 1)
Summary
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. The orthogonal IQ-Switch provides a state-ofthe-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms. The relatively high leakiness of Tet-On/Off[4] and the silencing of GAL4-responsive UAS element due to accumulated methylations on its CpG dinucleotides in vertebrates[5] have been potentially problematic the use of these systems in transgenesis. To overcome the methylation barrier in the GAL4/UAS-based system, a CpG-free element (tUAS) was adopted in zebrafish with a novel orthogonal driver (TrpR) but the unbearable toxicity of the driver limited its application in transgenesis[8]
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