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. SIMILAR CONTENT BEING VIEWED BY OTHERS DCAS9-VPR-MEDIATED TRANSCRIPTIONAL ACTIVATION OF FUNCTIONALLY EQUIVALENT GENES FOR GENE THERAPY Article 07 February 2022

REGULATED CONTROL OF GENE THERAPIES BY DRUG-INDUCED SPLICING Article 28 July 2021 MULTIPLEXED GENOME REGULATION IN VIVO WITH HYPER-EFFICIENT CAS12A Article 12 April 2022 INTRODUCTION Though

a variety of inducible transgene expression switches (ITESs) with binary features have made a substantial impact on scientific progress for understanding basic cellular functions of genes of

interest, discrete limitations of hitherto developed ITESs have been preventing their wider applications at the whole-organism level, in particular in vertebrates1,2. A common ITES is

composed of two elements. One is a driver construct, which encodes a promoter for the enforced expression of a stimulus-responsive heterologous transcriptional activator (TA), and the other

is an effector construct that consists of tandem repeats of TA binding elements followed by basal transcription-initiating sequences required for the transgene expression through the input

of an adequate stimulus. The stimulus can be a specific chemical or pleiotropic stress, including heat shock, heavy metals, or hormones. Among the various binary ITESs,

tetracycline-dependent transactivator (Tet-On/Off) and GAL4/UAS-based transgene switches have been most frequently used for the generation of genetic model organisms2. Especially, a

sophisticatedly designed heterologous GAL4-TA composed of an isolated GAL4 DNA-binding domain and a minimal VP16 activation domain fused to a modified ecdysone-binding region of EcR had been

adopted in zebrafish for the controlled expression of transgenes which could be tightly regulated by several different ecdysone agonists3. However, the relatively high leakiness of

Tet-On/Off4 and the silencing of GAL4-responsive UAS element due to accumulated methylations on its CpG dinucleotides in vertebrates5 have been potentially problematic the use of these

systems in transgenesis. The leakiness of ITESs could be completely plugged by concomitantly exploiting inducible Flp/_FRT_ or Cre/_LoxP_ DNA recombinase-based systems to initiate transgene

expression by the excision of regulatory elements6,7. However, as a DNA recombinase-based ITES becomes completely irreversible once stimulated, it may not be ideal when tunable or stepwise

transgene expression is preferable. 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 nonetheless, the unbearable toxicity of the driver limited its application in transgenesis8. Other currently available binary ITESs are valuable for the generation of transgenic

animals, however, each has also inherent drawbacks. For instance, the chemical stimulators mifepristone and rapamycin, which have been used in the LexPR-LexOP9,10, and dimerizer1,11 systems,

respectively, have potential adverse effects on reproduction and embryonic development. Moreover, as the dimerizer system becomes irreversible after treatment with rapamycin, it would not

be an optimal option if adjustable expression of transgene were necessary. To extricate the transgene expression switch from gene silencing, the QF/QUAS binary expression system has been

recently introduced to zebrafish transgenesis12,13,14. The Q system, which originated from a quinic acid-sensing system of _Neurospora crassa_, is composed of a TA (QF), five tandem repeats

of QF-responsive element (5xQUAS), and a QF repressor (QS)15,16. The QUAS element can be derepressed by the addition of quinic acid, which interferes with the inhibitory effect of QS on

QF16. Though the Q system has been shown to function in diverse organisms including _Escherichia coli_17, _Drosophila_16,18, _Caenorhabditis elegans_19,20, and plants21, several intrinsic

limitations of the system have hampered its direct application in vertebrates. These limitations include high toxicity of the QF driver12,13,14, non-functionality of quinic acid on QS in

mammalian cells, which prevents derepression by quinic acid16, and detrimental effects of quinic acid on zebrafish embryonic development12. However, despite the aforementioned drawbacks, the

potentially non-silencing feature of QF/QUAS would still be advantageous over the GAL4/UAS system12. Current efforts have been devoted to combining the Q system with other gene switches,

such as Tet-On20 and the bacterial Lac repressor system22, to circumvent the problematic traits of the original Q system. To expedite the progress of transgenesis in zebrafish, we newly

developed a more intuitive and reliable QF/QUAS-based orthogonal ITES, which is free of gene silencing at least up to seventh generations, equipped with a non-toxic driver, non-leaky, highly

tunable for expression of the transgene by an innocuous chemical inducer, reversible, and able to trigger several-fold higher transgene expression than the GAL4/UAS system. Hence, the

QF-based binary gene switch we report in this study overcomes pivotal problems of currently available ITESs, thus providing a valuable experimental platform for the regulated expression of

transgenes in zebrafish, with the potential for applications in cell-based systems and other whole organisms. RESULTS REFINING A NOVEL QF DRIVER THAT IS SENSITIVE TO EXOGENOUS CHEMICAL

STIMULI To apply Q-system-based ITESs in zebrafish transgenesis, we created various chimeric transactivators that are sensitive to chemical stimuli to drive luciferase expression under the

control of 5xQUAS. Initially we fused a hormone-binding domain of insect ecdysone receptor (EcR) to the full length QF TA at its C-terminus to gain nuclear access only when the chimera binds

to the ecdysone agonist, tebufenozide (Teb, also known as RH-5992), a lipophilic molecule that is non-toxic to developing zebrafish (Fig. 1a, b)3,23,24. The chimeric construct driven by the

zebrafish ubiquitin promoter25 was highly effective in inducing luciferase expression in HEK293 cells; nonetheless, it was too toxic to adopt for zebrafish transgenesis (Fig. 1a–c,

Supplementary Fig. 1 and Supplementary Table 1). Though the middle domain truncated form of QF was reported to be non-toxic in _Drosophila_26, it was still detrimental to zebrafish embryos

and unacceptably leaky (Fig. 1a–c, Supplementary Fig. 1 and Supplementary Table 1). Therefore, we modified the original QF until we obtained a construct that could suppress the intrinsic

toxicity while retaining the functional aspect as a TA. The QF is composed of a DNA-binding domain (QFDBD) at its N-terminus, followed by a minimal activation domain (AD*) that is regarded

as an N-terminal part of a middle domain (MD), and a main transcriptional activation domain (AD) (Fig. 1a) 26. Among the several driver constructs we tested, there was only one orthogonal

configuration selected for further assays. The selected driver was composed of a QFDBD, two tandem repeats of AD*, and a minimal VP16 transactivator (VP16*)3 followed by EcR

(QFDBD-2xAD*-VP16*-EcR, Driver 3 in Fig. 1a–c). In zebrafish embryos the driver did not elicit significant toxicity, irrespective of Teb treatments (Supplementary Figs. 1, 2 and

Supplementary Table 1). In addition, though relatively weaker than the original QF, the driver (hereafter IQ-Switch, Inducible QF transgene expression Switch) responded well to Teb without

gross luciferase leakiness (Fig. 1a–c and Supplementary Table 1). An alternative was a QF-Gal4 heterologous driver (QFDBD-Gal4AD), which was recently reported to show a tolerable toxicity

but capable of stimulating transgene located downstream of 5xQUAS in zebrafish14. However, in comparison with QFDBD-2xAD*-VP16*, QF-Gal4 was relatively more toxic in proportion to the amount

of transcripts whose expression elicited eccentric embryonic defects, including headless and severe ventralization, which were not observed with comparable injection of _QFDBD-2xAD*-VP16_*

mRNA (Supplementary Table 1 and Supplementary Fig. 2). The IQ-Switch translocated into the nucleus after administration of Teb, and then turned on the _mCherry_ reporter gene located after

5xQUAS in HEK293 cells (Supplementary Fig. 3a–j). The activity of the luciferase reporter was gradually potentiated by the increased dosage of Teb until it reached a plateau at 5 μM of Teb

in cell culture medium (Fig. 1d). The Teb dosage-dependent responsiveness of IQ-Switch was corroborated again in zebrafish embryos by western blotting. The IQ-Switch F2 driver,

_Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_), was crossed with the F2 effector, _Tg_(_13xQUAS:ZsGreen-P2A_), to obtain F3 siblings that were then exposed to different concentrations of Teb. Similar to

the cell culture results (Fig. 1d), we observed that ZsGreen expression levels gradually increased in proportion to the concentration of Teb (Supplementary Fig. 4). To test whether the

IQ-Switch was reversible by the withdrawal of the chemical inducer, we generated a transgenic zebrafish carrying a mutated _lamin A_ (_Δ37_) gene causative of Hutchinson-Gilford progeria

syndrome27,28. As expected, a genetic cross between the _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_) driver line (F2) and _Tg_(_5xQUAS:ZsGreen-P2A-lamin A__Δ37_) effector line (F3) produced embryos

reactive to Teb (50 μM) with expected Mendelian inheritance ratios, with ~25% of embryos showing ZsGreen expression. The ZsGreen-positive embryos were collected at the indicated time points

in Fig. 1e for the preparation of cDNA, which was then subjected to quantitative polymerase chain reaction (RT-qPCR) with primer sets specific for zebrafish _β-actin_ and _ZsGreen_. The

yield of transcripts encoding _ZsGreen-P2A-lamin A__Δ37_ was gradually reduced after Teb removal, indicating the reversibility of IQ-Switch (Fig. 1e). The relatively slow off-kinetics of Teb

corresponded well with the previous reports4,23,29 showing that it took time to completely metabolize ecdysteroids and their agonists in cells. In mice, the ecdysteroids are taken up and

metabolized by the liver before excretion through both urinary and fecal routes23. Another merit of IQ-Switch is that by removing EcR, the tunable transactivator becomes diverted into a

constitutively active form (hereafter EQ-On, Everlasting QF transgene switch-On), where its configuration no longer requires Teb stimulation for constant transgene induction (Driver 10, in

Fig. 1a). The EQ-On was mainly localized in the nucleus (Supplementary Fig. 3b, b′, k-r, k′-r′) and more than two-fold stronger than IQ-Switch (Fig. 1f) with Teb stimulation, with minimal

toxicity (Supplementary Figs. 1 and 2). The potential use of the switch was validated in zebrafish, in which the progeny obtained from the genetic cross of _Tg_(_ubb:QFDBD-2xAD*-VP16*_) and

_Tg_(_5xQUAS:ZsGreen-P2A-lamin A__Δ37_) showed ZsGreen reporter expression as early as 6 h post fertilization (hpf) without Teb administration (Supplementary Fig. 5). METHYLATION-INDEPENDENT

ACTIVATION OF IQ-SWITCH A previous report showed that the existence of multiple CpG dinucleotides in 5xQUAS did not interfere with GFP reporter expression by the stimulation of a QF-Gal4

heterologous driver14, and that artificial CpG-free 5xQUAS could not elicit further strong fluorescent reporter gene expression over the natural 5xQUAS14. Thus, we hypothesized that the

natural feature of CpG dinucleotides in 5xQUAS would be refractory to methylation. To identify the methylated state of the 5xQUAS element, we carried out bisulfite sequencing analysis of the

F4 generation of effector zebrafish having a human _BRAF__(V600E)_ transgene under the control of 5xQUAS, _Tg_(_5xQUAS_:_ZsGreen-P2A-BRAF__V600E_). As a control, we used the F2 effector

line with the zebrafish _braf_(_V610E_) transgene under the control of 10xUAS element, _Tg_(_10xUAS_:_ZsGreen-P2A-braf__V610E_), and _Tg_(_ubb_:_Gal4-VP16*-EcR_), which is responsive to a

Gal4 driver3 with Teb stimulus. Interestingly, methyl groups were heavily deposited on CpG dinucleotides in both elements (Fig. 2a), suggesting that activation of the 5xQUAS element occurs

irrespective of its methylation. To evaluate whether the IQ-Switch driver could bind to methylated QUAS to trigger transgene expression, we stimulated embryos obtained from genetic crosses

between driver and effector lines with 50 μM of Teb for only 2 h to elude early embryonic lethality of _braf__(V610E)_ and _BRAF__(V600E)_ transgenes30 (Fig. 2b, Supplementary Fig. 6a).

Indeed, enforced expression of drivers by injection of mRNA encoding _QFDBD-2xAD*-VP16*-EcR_ with a 6x_Myc_ epitope at its N-terminus into the _Tg_(_5xQUAS:ZsGreen-P2A-BRAF__V600E_) effector

(F5 generation) caused distinctive embryonic malformation when Teb (50 µM) was administered for the indicated time window (Fig. 2b, c). At the molecular level, through the chromatin

immunoprecipitation (ChIP) assay, we found that enrichment of the 6x_Myc_-tagged driver on the 5xQUAS elements, but uninjected or _6xMyc-Gal4DBD-VP16*-EcR_ mRNA-injected control embryos did

not elicit statistically significant levels of chromatin precipitation under identical experimental conditions (Fig. 2b, c). The enrichment of IQ-Switch driver on the methylated 5xQUAS was

not a unique feature of QFDBD-2xAD*-VP16*-EcR because we also observed a similar enrichment of 6xMyc-QFDBD-Gal4AD-EcR14 on the 5xQUAS under identical experimental settings with mRNA encoding

_6xMyc-QFDBD-Gal4AD-EcR_ instead of _6xMyc-QFDBD-2xAD*-VP16*-EcR_ (Supplementary Fig. 6). Therefore, the data strongly suggest that QFDBD binds to its specific cis-regulatory elements

irrespective of its methylation status. Moreover, while a genetic cross of F3 _Tg_(_ubb_:_QFDBD-2xAD*-VP16*-EcR_) driver and F6 _Tg_(_5xQUAS_:_ZsGreen-P2A-BRAF__V600E_) effector with

exposure of the embryos to Teb (50 µM) resulted in ubiquitous ZsGreen reporter expression and severe embryonic malformation in F7 progeny, mating F2 _Tg_(_ubb_:_Gal4DBD-VP16*-EcR_) driver

with F2 _Tg_(_10xUAS_:_ZsGreen-P2A-braf__V610E_) effector resulted in F3 embryos with variegated expression of reporter genes and relatively mild embryonic defects upon identical Teb

treatment (Fig. 2e). Other representative embryos were included in Supplementary Fig. 7. Collectively these data strongly suggest that the QF DNA-binding domain stimulates transgene

expression through direct binding to the QUAS element irrespective of its methylation state. CONTROLLED EXPRESSION OF BRAF(V600E) MANIFESTED DISCRETE RASOPATHY PATHOLOGIES AMELIORATED BY A

SELECTIVE BRAF INHIBITOR A germ line-transmitted animal that ubiquitously expresses BRAF(V600E) cannot be maintained due to the high toxicity of the _BRAF__(V600E)_ gain of function (GOF)

mutation. Though a Cre/LoxP-based conditional knock-in mouse model with the BRAF(V600E) oncogene has been available for more comprehensive studies of disease progression31, the

CMV-Cre-mediated ubiquitous expression of BRAF(V600E) caused prenatal lethality, which hampered the design of strategies for therapeutic approaches. To test whether IQ-Switch can be used as

a new drug-screening platform, controlled temporal expression of human _BRAF__(V600E)_ oncogene32 was carried out by a genetic cross between _Tg_(_ubb_:_QFDBD-2xAD*-VP16*-EcR_) and

_Tg_(_5xQUAS_:_ZsGreen-P2A-BRAF__V600E_), followed by a low dose (2.5 µM) of Teb treatment at 36 hpf for 24 h, with or without administration of vemurafenib (15 µM), an FDA approved specific

inhibitor of BRAF(V600E), for 36 h (Fig. 3a). As expected, triggering BRAF(V600E) expression at a later stage of zebrafish embryonic development was sufficient to induce visible

malignancies such as craniofacial deformities, cardiac malformation, and cutaneous abnormalities, all of which were reminiscent of cardio-facio-cutaneous (CFC) syndrome, a RASopathy commonly

caused by BRAF mutations33,34,35,36 (Fig. 3i–p and Supplementary Fig. 8a–j). However, early induction of BRAF(V600E) before gastrulation caused severe embryonic malignancy with truncated

posterior structure and compromised forebrain without noticeable eye structures at 24 hpf (Supplementary Fig. 8b, k, l). Strikingly, the CFC syndrome-like phenotypes by ectopic expression of

BRAF(V600E) were profoundly ameliorated by simultaneous treatment with vemurafenib (Fig. 3q–w). Our data strongly suggest that the GOF disease models generated by our IQ-Switch can be used

as a reliable and amenable drug-screening platform to help design more sophisticated therapeutic approaches. OPTIMIZATION OF QUAS TANDEM REPEATS TO AUGMENT TRANSGENE ACTIVITY Though we

demonstrated that IQ-Switch with 5xQUAS has significant advantages over other ITESs, its responsiveness to Teb was relatively weak compared to the Gal4DBD-VP16*-EcR/10xUAS system (Fig. 4a,

b)3. To improve sensitivity to Teb and to increase the transgene expression level of IQ-Switch, we initially tried to modify the driver construct by replacing its transcriptional activating

module with other more potent activators (Supplementary Table 1). However, all of our efforts were futile because all transcriptional activating modules tested other than

QFDBD-2xAD*-VP16*-EcR showed at least one fatal flaw rendering them inadequate for their application to transgenesis (Supplementary Table 1). Thus, instead of altering the driver, we

increased the length of the QUAS tandem repeats until a satisfying level of transgene expression was achieved by generating 5x, 9x, 13x, and 17xQUAS constructs (Fig. 4a and Supplementary

Fig. 9). The luciferase reporter activity in HEK293 cells was gradually improved by increasing the number of QUAS elements with Teb stimulation. The 13xQUAS together with the IQ-Switch

driver gave rise to strong _mCherry_ reporter gene expression in Cos7 cells when treated with Teb (Supplementary Fig. 3a′–j′), and its activity increased by 7.3-fold over the original 5xQUAS

(Fig. 4a, b). However, further increase of QUAS elements of up to 17 repeats failed to achieve stronger reporter activity, but rather compromised the luciferase activity on par with 9xQUAS

(Fig. 4a, b). Strikingly, the combination of QFDBD-2xAD*-VP16*-EcR with 13xQUAS was ~4.5 fold more potent than Gal4DBD-VP16*-EcR/10xUAS in the luciferase assay (Fig. 4a, b) with negligible

leakiness (Supplementary Fig. 10). The individual IQ-Switch with indicated combinations of QUAS repeats, with the exception of 17xQUAS, was adapted to zebrafish transgenesis (Supplementary

Fig. 11). The transgenic animals with ZsGreen reporter under the control of different tandem repeats of QUAS showed elevated expressivity of ZsGreen according to increased numbers of QUAS

elements when crossed with _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_) with a relatively low dosage of Teb (10 μM of Teb; Supplementary Fig. 11). To quantify transgene responsiveness to Teb (10 μM)

stimuli, we measured the fluorescence strength of the embryos obtained from genetic crosses between an F3 _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_) and discrete F2 effector lines with different

numbers of QUAS with a _ZsGreen_ reporter. To precisely measure the yield of fluorescence, we used a fixed confocal setting (see Materials for more detailed information) for the analysis of

fluorescence intensity in the brain and muscle, and background as a negative control (Fig. 4c). The fluorescent signals gradually increased in proportion to the number of QUAS repeats in

both the Z-stacking (Fig. 4c, d) and single-plane images (Supplementary Fig. 12). However, with high levels of Teb stimulation (50 μM of Teb), the strength of ZsGreen fluorescence at least

under the confocal microscope with 9xQUAS seemed to be reached a maximum level, comparable to that with 13xQUAS (Supplementary Fig. 13). Therefore, the expression level of IQ-Switch can be

modulated at least by two different approaches in vivo: via the dosage of the chemical inducer, and via the number of QUAS tandem repeats. The gradual increase in transgene expression

according to the order of QUAS repeats was quantitatively revalidated by western blotting in zebrafish embryos obtained from genetic crosses between an F3 _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_)

driver line with F2 effector lines, _Tg_(_5xQUAS:ZsGreen-P2A_), _Tg_(_9xQUAS:ZsGreen-P2A_), and _Tg_(_13xQUAS:ZsGreen-P2A_). The embryos from individual genetic crosses at 1 day post

fertilization (dpf) were subsequently treated with 10 μM of Teb for 24 h and then subjected to western blot analysis using anti-ZsGreen antibody. Teb-stimulated embryos under the control of

13xQUAS showed by far the most elevated level of ZsGreen expression in comparison to that with fewer QUAS tandem repeats (Supplementary Fig. 14). The different reactivities of the individual

QUAS constructs were validated again by observing transient ZsGreen reporter expression in the transgenic zebrafish _Tg_(_ubb:QFDBD-2xAD*-VP16*_) through introduction of plasmids encoding

discrete QUAS repeats upstream of _ZsGreen_. As expected, ZsGreen fluorescence was most intense under the control of 13xQUAS (Supplementary Fig. 15). Notably, the combination of IQ-Switch

with 13xQUAS showed an equivalent level of luciferase reporter activity to the other two potent driver configurations (QFDBD-2xAD*-VP32-EcR and QFDBD-Gal4AD-EcR), whose activities in general

reached the maximum intensity with 9xQUAS (Supplementary Fig. 16). NEURON-SPECIFIC EXPRESSION OF ZSGREEN WITH GRADUAL INTENSITIES USING IQ-SWITCH AND EQ-ON To test whether IQ-Switch was

applicable for labeling specific cells or tissues, we substituted ~8.7 kb of _elavl3_37 for the _ubb_ promoter in the driver cassette to visualize pan-neuronal tissues (Fig. 5 and

Supplementary Fig. 17). Importantly, genetic crosses between F1 _Tg_(_elavl3:QFDBD-2xAD*-VP16*-EcR_) and F2 _Tg_ with _ZsGreen_ transgene under the control of different numbers of QUAS (5x,

9x, 13x) activated ZsGreen expression with different strengths in the order of the length of QUAS repeats only when stimulated with 50 µM of Teb for 48 h from 3 dpf (Fig. 5a, b). When the

offspring obtained from a similar genetic cross between a Teb-responsive driver and _Tg(5xQUAS:ZsGreen-P2A)_ were exposed to the same dose of Teb for 24 h before imaging at 48 hpf, ZsGreen

fluorescence was observed throughout the entire neuronal tissues, including axonal fibers innervating neuromasts, which completely recapitulates the well-known _elavl3_ expression

domains38,39 (Supplementary Fig. 17). Even more striking was the EQ-On dependent expression of ZsGreen in neurons driven by the cross between _Tg_(_elavl3:QFDBD-2xAD*-VP16*_) and

_Tg(QUAS:ZsGreen)_ in which fluorescent signals were gradually rising in whole neurons, including sensory neurons in the caudal fin where the ZsGreen became most intensified by far with

13xQUAS (Fig. 5c, I-III). As the caudal fin axon branches are not usually observed in conventional _Tg_(_elavl3:EGFP)_38, our modified QF/QUAS transgene induction systems could be used to

unveil weak expression domains by potentiating the promoter activity. The process of neuronal development of growing embryos can be analyzed by labeling neurons with appropriate fluorescent

markers. Therefore, we set out to trace developing sensory neurons in epithelial cell layers in the yolk (ECL) for three days starting from 2 dpf whose sensory neurons have been analyzed

with limited success due to the lack of adequate molecular and genetic tools to label the neurons in ECL in zebrafish embryo40. As shown in Fig. 6a and Supplementary Fig. 18, the gradual

increase in fluorescent signals in anastomosing networks in ECL across developmental procedures became evident in embryos with 13xQUAS:ZsGreen at 5 dpf in comparison to that of

5xQUAS:ZsGreen. Given the mesh-like appearance of the fluorescent signals in the yolk (Supplementary Fig. 18) and the equivalent background noise between 5xQUAS:ZsGreen and 13xQUAS:ZsGreen

clutches, the observed fluorescence in ECL could not be simply due to the advent of auto-fluorescence or different imaging settings (Fig. 6b). In addition, to minimize the possible emergence

of auto-fluorescence in the yolk, we used a fixed confocal setting that did not elicit any perceivable auto-fluorescence when observing the embryos. Taken together, IQ-Switch should be able

to tune transgene expression in a spatiotemporal manner via combining different drivers and effectors. In addition, the EQ-On would make enforced reporter gene expression possible even with

a relatively weak promoter. DISCUSSION Although there are miscellaneous binary transgene induction cassettes available, one or more inherent weaknesses have hampered their application for

the generation of transgenic animals. For instance, Tet-On/Off and stress-inducible promoters are not recommended for conditional gene expression due to the relatively high leakiness of the

transgene4,41. The GAL4/UAS system has been most successfully applied to zebrafish in transgenesis as well as in enhancer trapping42,43 owing to its many advantages including a tolerable

degree of driver toxicity, low leakiness, reversibility, and a relatively high expression level of transgenes. Furthermore, the versatile heterologous Gal4 drivers conjugated with distinct

regulatory modules responsive to their compatible stimuli such as drugs, and the vast depository of transgenic lines under the control of UAS that can be combined by genetic crosses with the

discrete Gal4 driver provide a great resource for generating new transgenic zebrafish3,44,45. However, cumulative methylation of the UAS element after successive generations results in

mosaicism of transgene expression as early as the F3 generation (Fig. 2e) that may eventually attain complete transcriptional silencing5. The methylation-driven transcriptional silencing of

transgenes could be overcome by adopting an ITES without containing CpG dinucleotides in TA-responsive elements as TrpR/tUAS8 or using a GAL4/UAS system with a limited copy of UAS tandem

repeats5,46. Although TrpR/tUAS has the virtue of being free from transgene silencing8, it is regarded as barely suitable for transgenesis without substantial alleviation of the toxic

features of TrpR drivers. As previously reported, four copies of the non-repeating UAS construct (4Xnr UAS) were less susceptible to methylation, whereby GAL4 binding properties seem to be

retained after three generations5. The refractory feature of the Q system against transgene silencing across successive generations12,13 has drawn much attention from researchers developing

new molecular and genetic tools to tackle technical challenges such as methylation-driven promoter inactivation. However, the original Q system12 could not be adopted as an alternative for

generating transgenic zebrafish because of the substantial driver toxicities as shown in Supplementary Fig. 1. Although another QF driver (QF-Gal4) was recently developed as an additional

gene switch14, the potential toxicity of the driver, at least under our experimental conditions, may limit its usage for zebrafish transgenesis (Supplementary Fig. 2); a reliable

QF/QUAS-based gene-tunable cassette suited to vertebrate systems without toxicity has not yet been developed. Therefore, a novel transgene switch with no gene silencing problem and minimal

toxicity while retaining all the virtues of the QF/QUAS binary ITES is highly desirable. To satisfy the aforementioned unmet needs of ITESs, we developed a new binary transgene cassette

designated as IQ-Switch, which meets all of the prerequisite criteria for its use in vertebrate models: first, it shows no discernable toxicity or transgene leakiness; second, it shows a

several-fold higher responsiveness to the non-toxic chemical inducer Teb compared to the GAL4/UAS system; third, it does not become silenced after its transmission to the next generation,

irrespective of its promoter methylation; fourth, the expression level of the transgene can be easily manipulated by altering concentrations of the chemical inducer, or by exploiting

effector lines having different numbers of QUAS element tandem repeats; fifth, by using tissue specific promoters, researchers are able to induce the gene of interest in a spatial and

temporal manner; sixth, the system is reversible by withdrawal of the chemical inducer; seventh, using a driver that does not have an EcR domain (EQ-On), one can drive transgene expression

constitutively by a simple genetic cross with an appropriate effector line without treatment with chemical inducers; eighth, individual effector constructs with different numbers of QUAS

repeats could be combined with other potential drivers, for instance QF-Gal414, to modulate the yield of transgene expression depending on experimental purposes; and ninth, as a genetic

cross with 13xQUAS effector highly potentiated the driver’s promoter activity (Fig. 5c), our newly developed gene switch could be applied to transgenesis for the identification of a given

promoter’s activity in tissues where it is too weak to demarcate itself. One potential concern of the IQ-Switch is the possible adverse effects of ecdysone analogs on cellular

physiology47,48,49. High concentrations of Teb (over 142 μM) induced cell cycle arrest and apoptosis in the human cervical carcinoma cell line HeLa48,49; other ecdysone agonists including

muristerone A, ponasterone A, and GETM-E inhibited FasL-and TRAIL-induced cell death in the human colon carcinoma cell line RKO47. We used Teb at no more than 50 μM in zebrafish embryos;

this concentration appears permissive to early embryonic development; however, we could not completely rule out the possibility that Teb concentration might be deleterious to the transgenic

animals when they are raised with Teb for a prolonged period. Therefore, it would be desirable to substitute Teb with other ecdysone analogs with less or no cellular toxicity. In addition,

for expanding IQ-Switch in broader biological applications, generating a singular plasmid by combining all the requisite components of the driver and effector of IQ-Switch would be helpful,

as was the case with a Gal4/UAS-based singular gene switch4. The relatively low transactivating activity of IQ-Switch may compare to QF-Gal4 (Supplementary Fig. 16); it appears that although

the transactivating activity of QF-Gal4-EcR is similar or higher with Teb treatment than IQ-Switch, and that of QF-Gal4 is higher than that of EQ-On (Supplementary Fig. 16c), QF-Gal4 is

more toxic, especially when it is strongly expressed (Supplementary Fig. 2). Thus, IQ-Switch might be a reliable ITES that can balance a relatively high transactivation activity and

restrained toxicity. Therefore, IQ-Switch is a beneficial transgene-inducible cassette that can overcome the intrinsic obstacles frequently observed in currently available ITESs. In

combination with other gene switches including Gal4/UAS5,46 and LexPR-LexOP9,10, the IQ-Switch can serve as an additional and complementary genetic tool to currently available ITESs for the

preclinical studies of GOF diseases and for elucidating the cellular and embryonic function of genes of interest. In addition, our data strongly suggest that IQ-Switch as well as EQ-On can

be applied to other model organisms for the precise control of transgene expression. METHODS PREPARATION OF PLASMIDS FOR TRANSGENESIS A Tol2 vector (pKY64-miniTol2-opt-CRY-eFGP) was used as

a prime backbone of the plasmid construction (Supplementary Table 2). Individual elements, promoters and all the required elements for generating transgene expression cassette were subcloned

into the Tol2 vector (pKY64-miniTol2-opt-CRY-eFGP) using T4 DNA polymerase (NEB)-mediated ligation-independent cloning method following the experimental procedures in a previous report50.

The _elavl3_ promoter from pTol2-elavl3-GCaMP6s (Addgene plasmid #59531) was a gift from Dr. Misha Ahrens. All of the vectors we generated are shown in Supplementary Table 2. ZEBRAFISH

HUSBANDRY AND TOL2 MEDIATED TRANSGENESIS Zebrafish (_Danio rerio_, AB line) were raised at 28.5 °C with proper water circulation and maintained under a light schedule of 14 h light and 10 h

dark. For the generation of transgenic zebrafish, 50 pg of transposase mRNA51 together with respective plasmids (20 pg each) listed in Supplementary Table 2 were pressure injected at 1-2

cell stages. The in vitro transcribed mRNA encoding transposon52 was synthesized using mMESSAGE mMACHINETM SP6 transcription kit (Thermo Fisher Scientific). Among the injected embryos,

fluorescent positive siblings in lens at 4 dpf were collected for the husbandry. After one to one outcross of the founder fish listed in Supplementary Table 3 with a wild type AB line, the

siblings positive of EGFP or mCherry fluorescence in lens were collected and raised to adulthood. Three independent lines of each transgenic animal have been maintaining through consecutive

outcross with wild type AB line. Zebrafish care was performed in accordance with guidelines from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and Chungnam National

University (201903-CNU-007). All of the transgenic animals we generated are shown in Supplementary Table 3. GENOMIC DNA ISOLATION AND BISULFITE SEQUENCING

_Tg_(_10xUAS:ZsGreen-P2A-braf__V610E__, α-cry:EGFP_) and _Tg_(_5xQUAS:ZsGreen-P2A-BRAF__V600E__, α-cry:mCherry_) thirty embryos each at 7dpf were randomly collected and homogenized

uniformly. The genomic DNA was isolated using TIANamp Genomic DNA Kit (TIANGEN) following the manufacturer’s instruction. Bisulfite sequencing was performed targeting _10xUAS_ or _5xQUAS_

sequences through company BIONICS.Inc (Korea). TOTAL RNA EXTRACTION AND CDNA SYNTHESIS For the preparation of total RNA, 30 embryos of each sample were randomly collected at the indicated

time points (Fig. 1e). 20 μl of Tri-reagent (Ambion) per embryo was added before homogenized using a 3 ml disposable syringe, and then 1/5 volume of chloroform was added to each sample. The

stirred samples were centrifuged (13,000 RPM, 10 min at 4 °C), and then the supernatant was transferred into fresh tube. Total RNAs were precipitated with ice-cold isopropyl alcohol. For the

cDNA synthesis, 2.5 μg of total RNA was used for the in vitro reaction with SuperScript reverse transcriptase II (Invitrogen) in accordance with the manufacturer’s instructions. IN VITRO

TRANSCRIPTION For the synthesis of mRNA, pCS2+ and pCS2+MT vectors were exploited for the delivery of proper DNA. The vectors were linearized by the digestion with Not I, and then cleared

out using a phenol/chloroform extraction. The 1 μg of linearized template were subjected to in vitro reaction using mMESSAGE mMACHINE™ SP6 Transcription Kit (Invitrogen) following the

manufacturer’s instruction. The in vitro transcribed mRNA was dissolved in 0.1 M DEPC treated KCl before injection. CHROMATIN IMMUNOPRECIPITATION (CHIP) In vitro synthesized mRNAs encoding

indicated drivers with N-terminal 6xMyc tag were pressure injected into the progeny from _Tg_(_5xQUAS:ZsGreen-P2A-BRAF__V600E__, α-cry:mCherry_). Embryos at 20-somite stage were fixed with

2.2% paraformaldehyde in egg water (60 μg of sea salt/ml) for 30 min, and then quenched under the 0.125 M glycine. The prepared embryos were homogenized using a disposable syringe in IP

buffer (150 mM NaCl, 10 mM Tris-Cl pH 7.5, 1 mM DTT and 0.2% NP40). The embryo lysates were centrifuged (13,000 RPM, 1 min) to obtain nuclear pellets which were subjected to sonication for

the preparation of sheared genomic DNA. While 10% of sheared DNA was secured as input control, the rest of the sample was used for ChIP through the incubation with 1 μg of monoclonal

anti-Flag (M2, Sigma-Aldrich) or 1 μg of mixtures of anti-Myc antibodies (Santa Cruz Biotechnology, sc-40 plus Millipore, 06-549) for 3 h at 4 °C. The DNA fragments bound by Myc-tagged

proteins were obtained using Chelex 100 resin (Bio-Rad) following a protocol53,54 with a small modification. The amount of precipitated DNA was analyzed using qPCR. QUANTITATIVE PCR (QPCR)

For the quantification of the yield of transgene and immunoprecipitated DNA, a TOPreal™ qPCR 2x PreMIX (Enzynomics, Korea) was used for the measurement of amplified samples by exploiting a

CFX connect Real-Time PCR Detection System (Bio-Rad) with Bio-Rad CFX Manager™ 3.1 software for the calibration. The yield of _ZsGreen_ was measured in comparison to that of _β-actin_. The

assays in Figs. 1e and 2c were carried out in triplicates and error bars stands for the standard error. The calculated data were revalidated using Microsoft Excel. The statistical

significance depicted as _p_-value was represented in Fig. 2c. Only the _p_-value less than 0.05 considered as statistically significant. The following primer sets were used for the

detection of the yield of individual samples: A ZsGreen primer set, for the amplification of 88 bp length of DNA fragment: _ZsGreen_ forward primer, 5′-CATCTTGAAGGGCGACGTGAG-3′; _ZsGreen_

reverse primer, 5′-CTTGGCCTTGTACACGGTGTC-3′; A _β-actin_ primer set, for the amplification of 120 bp length of DNA fragment: _β-actin_ forward primer, 5′-TACAATGAGCTCCGTGTTGCC-3′; _β-actin_

reverse primer, 5′-AGGGGTGTTGAAGGTCTCGAA-3′; A primer set for targeting QUAS element to amplify 231 bp of DNA fragment: QUAS amplicon forward primer, 5′-CGAGGTCGACAACTTTGTATAGAAAAGTTG-3′;

QUAS amplicon reverse primer, 5′-TACAAACTTGACGCGTCTTCGAGG-3′. TEBUFENOZIDE AND VEMURAFENIB TREATMENT Tebufenozide (Sigma-Aldrich, 31652) was dissolved in dimethyl sulfoxide (DMSO) at 50 mM

stock solution, which is further diluted before treatment to cells or zebrafish embryos in proper culture media. To remove tebufenozide, embryos were washed out several times with fresh egg

water (60 μg of sea salt/ml). The vemurafenib (PLX4032; S1267) was purchased from Selleckchem.com (U.S.). Adequate amount of vemurafenib dissolved in DMSO was directly added to the embryos.

LUCIFERASE ASSAY The 5x, 9x, 13x, 17xQUAS and 10xUAS were subcloned into pGL3-Basic vector (Promega). HEK293 cells that were 50% confluent were transfected with 40 ng of pRL-CMV (Promega)

together with each 200 ng of driver and effector plasmids using GENE-FectTM transfection reagent (TransLab Inc, Korea). Luciferase assays were carried out using Dual-Luciferase Reporter

Assay System (Promega) following the manufacture’s instruction. Each sample was measured at least three times, and significance was assessed by the Student’s _t_ test. Briefly, the

transfectants were cultured for 5 h before treatment of Teb with indicated amount, and then harvested for dual-luciferase assay after further extended culture for another 24 h The

statistical significance was represented in Fig. 1f with _p_-value. For _p_-value >0.05, we consider the difference not significant. The standard deviation and Student’s _t_ test

_p_-value were obtained through exploiting Microsoft Excel. QUANTIFICATION OF FLUORESCENCE IMAGING OF ZEBRAFISH TRANSGENIC ANIMALS To quantify the relative fluorescence intensity of confocal

images with different lengths of QUAS tandem repeats, confocal images of the progeny were obtained from genetic crosses of _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_) with transgenic lines integrated

with 5x, 9x, and 13x QUAS _ZsGreen_ reporter in their genome at 2 dpf using the FV 1000 confocal microscope (Olympus). Before confocal imaging, transgenic zebrafish embryos were incubated

with Teb (10 μM) at 1 dpf for 24 h. Zebrafish embryos from the cross of _Tg_(_ubb:QFDBD-2xAD*-VP16*-EcR_) and Tg with a 13xQUAS _ZsGreen_ reporter without Teb incubation were used as

negative controls. Quantitative analyses were performed by measuring the relative fluorescence intensity using Image J with the ROI manager (https://imagej.nih.gov/ij/) as previously

described55. Briefly, Z-projected confocal images were generated by stacking 10 sections with 10 μm thickness. To precisely measure the fluorescence yield, regions of Z-projected images were

first divided into three parts (background, brain, and muscle areas), and then the relative fluorescence intensities of the background areas from different samples obtained by Image J with

the ROI manager were averaged and set as a basal level (A. U. = 1). Finally, the brain and muscle areas were chosen and the fluorescence intensities of ZsGreen with different tandem repeats

of QUAS, obtained by Image J with the ROI manager, relative to the average basal level, were calculated. Notably, the initial relative fluorescence intensities of the background areas of

different groups were very similar to each other, confirming that the images were taken under identical conditions (Fig. 4c). Although this comparative analysis was sufficient to reveal the

differences among different samples and areas, the fluorescence of the brain area of the 13xQUAS _Zsgreen_ reporter line, which was the strongest, tended to be saturated using the current

imaging parameters. To circumvent this fluorescence saturation issue, we also measured the fluorescence intensities at single planes of confocal Z-stack images (Supplementary Fig. 12). We

chose a single optical section that exhibited the strongest fluorescence intensity out of the total of 10 optical slices of each Z-stack that usually fell into one of the 4th–6th slices. In

the case of the negative control without Teb, the 5th single plane was selected and its fluorescence intensities in different areas were measured in the same way as described above. Twenty

transgenic zebrafish at 2 dpf per group were measured for quantitative analysis. To show the development of axon tracks in the yolk (Fig. 6 and Supplementary Fig. 18),

_Tg(elavl3:QFDBD-2AD*-VP16*)_ zebrafish embryos with 5x or 13xQUAS _ZsGreen_ reporter were used for imaging. Representative images of embryos at developmental stages from 2 dpf to 5 dpf were

obtained daily under the same experimental conditions. To show that images from experiments were taken under identical confocal imaging conditions, the relative fluorescence intensities of

the background area of zebrafish embryos with 5x or 13x QUAS _ZsGreen_ reporter lines were obtained using Image J with ROI manager as described above and compared. Statistical analyses of

the data were performed using the Student’s _t_ test. The statistical significance is represented in Figs. 4d, 6b and Supplementary Fig. 12, with _p-_values. _A p_-value higher than 0.05 was

considered not significant. The standard deviation and Student’s _t_ test _p_-values were obtained using Microsoft Excel. Fluorescence Z-stack images in Fig. 3 were also obtained by using a

CELENA®S digital imaging system (Logos biosystems, Korea). FLUORESCENCE IMAGING OF CELLS After transfection of various combination of driver and effector plasmids with appropriate Teb

stimuli (10 μM, for 12 h) if necessary, HEK293 and Cos7 transfectants on coverslips were washed three times with PBS and then fixed with 4% paraformaldehyde for 15 min. Nuclei were stained

with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542) for 2 min. After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (TCS SP8; Leica,

Wetzlar, Germany), with constant excitation, emission, pinhole, and exposure time. Equipment parameters were summarized in Supplementary Table 4. WESTERN BLOTTING After 30 dechorionated

zebrafish embryos were rinsed with ice-cold PBS for three times, they were lysed by trituration with a 3 ml disposable syringe in 10 μl of CompLysisTM Protein Extraction Reagent (SignaGen

Laboratory, SL100319-L) per embryo with ProEXTM 1x protease and phosphatase inhibitor cocktail (TransLab Inc, Korea, TLP-120I). The lysates were incubated for 15 min on ice, they were

subjected to centrifugation for 10 min at 16,000 × _g_ at 4 °C, and then the supernatant was mixed with 5x SDS gel loading buffer before boiling for 5 min. The 12.5 μl of each sample with

SDS gel loading buffer being equivalent to the amount of protein extracted from a single embryo was separated in 10% SDS-PAGE, and then transferred into nitrocellulose membrane, 0.45 μm

(Pall Corporation). The membrane was immersed in 1x blocking solution (ProNATM 5x phospho-Block Solution, TransLab Inc, Korea, TLP-115.1 P) at room temperature (RT) for 30 min, incubated

with primary antibodies diluted in 1x blocking solution for 8 h at RT. After the membrane was rinsed three times with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 8.0),

HRP-conjugated secondary antibody diluted in 1x blocking solution was added and then incubated for 1 h at RT. After vigorous washing the membrane with TBST over 5 times, western detection

was performed using an ECL detection system supplied from TransLab (ProNATM ECL Ottimo, TLP-112.1) under the chemiluminescence CCD imaging system (ATTO, Ez-Capture MG). Following antibodies

were used in this assay; anti-ZsGreen (Takara Bio Clontech, 632474), anti-actin (Sigma-Aldrich, A2066), goat anti-rabbit IgG secondary antibody-HRP (Thermo Fisher Scientific, 31460).

STATISTICS AND REPRODUCIBILITY All the luciferase assays and qPCR measurements were carried out triplicate or more. In the case of western blotting, three independent experiments had been

analyzed using Image J before calculating _p_-value. Only the calculated _p_-value less than 0.05 considered as statistically valid. ZsGreen fluorescence intensity in zebrafish embryos using

Image J was calibrated in comparison with their own background intensity. The number of embryos analyzed was depicted in appropriate figure legends. REPORTING SUMMARY Further information on

research design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The raw data for graphs is available in supplementary data 1. Any further

information can be obtained from corresponding authors upon reasonable request. The sequence information of the core vectors used in this study is shown in supplementary data 2, and the

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(Hoboken)_ 296, 378–381 (2013). Article  Google Scholar  Download references ACKNOWLEDGEMENTS The authors really appreciate Dr. Igor B. Dawid (NIH-NICHD, US) for the critical reading and

valuable comments on the manuscript. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2016R1D1A3B01010007,

No.2020R1A2C2005317, NRF-2019R1A2C1087934, and NRF-2019R1A2C1010661), by National Research Council of Science & Technology (NST) of Ministry of Science and ICT of Korea (CRC-15-04-KIST),

and by KRIBB Research Initiative Program (KGM5352113, KGM2112133). AUTHOR INFORMATION Author notes * These authors contributed equally: Jeongkwan Hong, Jae-Geun Lee, Kyung-Cheol Sohn.

AUTHORS AND AFFILIATIONS * Department of Biological Science, College of Biosciences and Biotechnology, Chungnam National University, Daejeon, Republic of Korea Jeongkwan Hong, Kayoung Lee, 

Seoee Lee, Jinyoung Lee, Jihye Hong, Dongju Choi, Yeseul Hong, Jangham Jung & Hyunju Ro * Disease Target Structure Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon, 34141,

Republic of Korea Jae-Geun Lee & Jeong-Soo Lee * Department of Functional Genomics, KRIBB School, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113,

Republic of Korea Jae-Geun Lee & Jeong-Soo Lee * Dementia DTC R&D Convergence Program, KIST, Hwarang-ro 14 gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea Jae-Geun Lee & 

Jeong-Soo Lee * Department of Pharmaclogy, College of Medicine, Chungnam National University, Daejeon, Republic of Korea Kyung-Cheol Sohn & Gang Min Hur * Biomedical Research Institute,

Chungnam National University Hospital, 282, Munhwa-ro, Jung-gu, Daejeon, 35015, Republic of Korea Hyo Sun Jin & Dae-Kyoung Choi * Natural Medicine Research Center, Korea Research

Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Republic of Korea Su Ui Lee * Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University,

Chuncheon, Republic of Korea Yun Kee * Division of Cancer Biology, Research Institute, National Cancer Center, Goyang-si, Republic of Korea Young-Ki Bae * Department of Nursing, Gwangju

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for this author inPubMed Google Scholar * Jangham Jung View author publications You can also search for this author inPubMed Google Scholar * Young-Ki Bae View author publications You can

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publications You can also search for this author inPubMed Google Scholar * Jeong-Soo Lee View author publications You can also search for this author inPubMed Google Scholar * Hyunju Ro View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS J.H., J.-G.L., and K.-C.S. equally contributed to the paper by carrying out construction of the

plasmids, generation of transgenic animals, ChIP, qPCR, western blotting, luciferase assays, data processing and confocal imaging of zebrafish embryos. K.L. also contributed heavily to the

construction of the plasmids and the generation of transgenic animals. H.S.J., and D.-K.C. took confocal images of animal cells. J.L., J.H., D.C., and Y.H. were involved in generation of

transgenic zebrafish carrying a mutated _lamin A_ (_Δ37_). S.L., S.U.L., Y.K., J.J., Y.-K.B., and R.H.H. contributed to the construction of plasmids and the maintenance of transgenic

animals. G.M.H., J.-S.L., and H.R. are co-corresponding authors who had supervised the whole process of the experiments and had written the manuscript. CORRESPONDING AUTHORS Correspondence

to Gang Min Hur, Jeong-Soo Lee or Hyunju Ro. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW INFORMATION _Communications Biology_ thanks Fumi

Kubo and Koichi Kawakami for their contribution to the peer review of this work. Primary Handling Editors: Jun Wei Pek and Anam Akhtar. Peer reviewer reports are available. ADDITIONAL

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hong, J., Lee, JG., Sohn, KC. _et al._ IQ-Switch is a QF-based innocuous,

silencing-free, and inducible gene switch system in zebrafish. _Commun Biol_ 4, 1405 (2021). https://doi.org/10.1038/s42003-021-02923-3 Download citation * Received: 28 May 2021 * Accepted:

24 November 2021 * Published: 16 December 2021 * DOI: https://doi.org/10.1038/s42003-021-02923-3 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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