IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish

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.

All-in-one IQ toggle switches with high versatilities for fine-tuning of transgene expression in mammalian cells and tissues

“…To address the flaws of EUI gene switch, we attempted to integrate the binary IQ-Switch, originally optimized for zebrafish transgenesis, 34 with several modifications. To create a novel SIQ, we assembled all the necessary elements for IQ-based transgene toggling into a pENTR-EUI vector.…”

“…Since the native QF transcriptional activator was highly toxic in fruit flies and zebrafish, 28 , 29 , 30 , 34 the QF had to be extensively amended before being adopted in animals to eliminate its cellular toxicity. We previously minimized the cytotoxicity issues of QF by removing virulent modules from the middle domain to the C-terminal end of large transactivation domain.…”

“…Recently, we reported a more reliable binary Q switch, IQ-Switch, optimized for the transgenesis of zebrafish. 34 The IQ-Switch showed almost undetectable leakiness of transgene expression, eliciting at least 4.5-fold more sensitivity to a chemical inducer Teb in comparison to Gal4/UAS under identical experimental conditions in zebrafish. 34 …”

Non-Mammalian Models for Understanding Neurological Defects in RASopathies

“…Expression of the common kinase-activating variant in CFC syndrome BRAF Q257R in zebrafish embryos increased the major/minor axis ratio [104]. Also, overexpression of BRAF V600E before gastrulation in zebrafish embryos caused severe embryonic malignancy with truncated posterior structure and compromised forebrain, while later induction led to craniofacial deformities resembling CFC syndrome [105] (Table S2). Activating mutations in MEK1 have different strengths, which are correlated with the severity of the phenotypes observed in human patients affected by these mutations [106].…”

“…In zebrafish embryos, studies have shown that expression of BRAF Q257R increased the major/minor axis ratio, a phenotype that was prevented by treatment with the MEK inhibitors CI-1040 and PD0325901 [104,155]. Similarly, the CFC-syndrome-like phenotypes associated with ectopic expression of BRAF V600E were profoundly ameliorated by simultaneous treatment with vemurafenib [105]. Furthermore, in the zebrafish model of NF1, treatment with sunitinib, a RTK inhibitor, effectively arrested the progression of transplanted MPNSTs, and combination treatment with both sunitinib and trametinib, a MEK inhibitor, enhanced this therapeutic effect [87].…”