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

Abstract

The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

Keywords: SIQ; SIQmate; adenovirus; cumate; phiC31; tebufenozide; transgene toggling.

Graphical abstract

Figure 1

A schematic diagram of SIQ transgene toggle switch (A) The pSIQ vector encompasses all the components of an IQ-Switch, including an IQ-Driver: QFDBD, two minimal activation domains of QF (2×AD∗), a minimal VP16 activation domain (VP16∗), and an EcR. These components are governed by a CMV promoter and required for binding to the 13× QUAS enhancer elements, subsequently inducing transcription of genes of interest only when stimulated by Teb. A phiC31 serine integrase recombination site is denoted as attB, where the recombination event occurs to the corresponding attP site in the presence of the recombinase. For ease of cloning into the pAd/PL-DEST destination vector, we included gateway cloning-associated elements (attL1/attL2) for the production of adenovirus. The kanamycin and puromycin antibiotic-resistant markers are denoted as KanaR and PuroR, respectively. (B) The all-in-one SIQ transgene toggle switch exhibits high versatility. With a single construct, a broad range of tailored transgene induction experiments can be carried out through transient transfection, generation of stable cell lines, and adenovirus-mediated transgene delivery. The arrows indicate that discrete purposes of experiments could be merged into a SIQ.

Figure 2

SIQ surpasses the Gal4/UAS-based singular gene switch regarding sensitivity and the issue of leakiness (A) The pEUI(+)-EGFP refers to a previously reported singular gene switch based on the Gal4/UAS system. The gradually elevated induction of the EGFP reporter, dependent on Teb dosage, was visualized under a fluorescence microscope after transiently transfecting 1 μg of the indicated plasmid constructs. After 24 h of transfection, Teb was treated for 24 h, and then the expression level of EGFP was observed using the same exposure times and contrasts. Scale bar, 200 μm. (B) Quantitative measurements of transgene expression levels were performed using the dual-luciferase assay. HEK293 cells were transfected with either pEUI(+)-Luciferase or pSIQ-Luciferase. After transfection, the cells were treated with Teb for 8 h. Statistical significance was analyzed using two-way ANOVA with Šidák correction (n = 3). ns, not significant. (C) The basal leakiness of pEUI(+) and SIQ was compared through a dual luciferase assay in the absence of Teb. Statistical significance was explored using the Student t test. The data are represented as mean ± SD (n = 3). ∗∗∗p < 0.001. A.U., arbitrary unit; Luc, luciferase.

Figure 3

Stable cell line harboring the SIQ system could be generated by integrating it into a specific locus of the host cell genome (A) A schematic diagram illustrating the phiC31-mediated genomic integration of the SIQ toggle switch. The phiC31 serine integrase targets the attB element of pSIQ and an attP site in the host cell genome, mediating directional recombination, resulting in producing attR and attL recombined sequences. (B) A representative result of genomic PCR using the primers depicted with red and black arrows in (A). The first PCR products amplified by primers labeled with red arrows were used for the second PCR reaction with primers depicted by black arrows. The expected size of PCR products was shown in (A) under the square brackets. c, control group with mock cell genome for PCR template; e, experimental group. (C) Established stable cell lines harboring SIQ-EGFP were exquisitely sensitive to Teb stimuli. EGFP was detected only in the group of cells treated with 10 μM Teb for 24 h. Bright field images were located on the left side. Scale bar, 200 μm. (D) EGFP was detected as early as 3 h after 10 μM of Teb treatment, as shown by western blotting. Endogenous expression of α-tubulin was used as a loading control. (E) After 24 h of Teb (10 μM) treatment, the cells were reseeded into fresh culture dishes to minimize Teb presence. The level of EGFP transcripts was measured by qRT-PCR after harvesting the cells at the indicated time points. The data are presented as mean ± SD. Scale bar, 200 μm. Statistical analysis was performed using One-way ANOVA with Tukey’s HSD test (n = 3). ∗∗∗p < 0.001. (F) Schematic comparison between pSIQ-EGFP without IQ-Driver (W/O Driver) and pSIQ-EGFP constructs. (G) FACS analysis of Mock (containing only an attP in their genome), SIQ-EGFP W/O Driver, and SIQ-EGFP stable cell lines after 24-h exposure to Teb (10 μM). Data are presented as mean ± SD (n = 3).

Figure 4

SIQ toggle switch was free from transgene leakiness (A) Bright field and GFP channel images of an established cell line harboring SIQ-EGFP-P2A-NTR 2.0 at the attP locus of HEK293 cells after 24 h of treatment with the indicated combinations of chemicals. Scale bar, 200 μm. (B) Measurement of caspase activities in the stable cell line harboring SIQ-EGFP-P2A-NTR 2.0 after 24 h of treatment with the indicated chemicals. Statistical analysis was performed using ordinary One-way ANOVA with Tukey’s HSD test (n = 3). The data are presented as mean ± SD. ∗∗∗p < 0.001.

Figure 5

An adenoviral SIQ toggle switch induced strong transgene expression in HaCaT cells and mouse liver (A) Direct comparison of adenoviral Gal4/UAS (Ad/EUI-EGFP) and SIQ (Ad/SIQ-EGFP) in HaCaT keratinocytes. Cells were infected with adenovirus at the indicated MOI and subsequently exposed to 10 μM Teb. Bright field images are shown above the GFP channel images. Scale bar, 200 μm. (B) Revalidation of fluorescence data from (A) by western blot analysis using an anti-EGFP antibody. Endogenous expression of β-actin served as the loading control. (C) In vivo analysis of EGFP expression in the liver after intravenous injection of increased Ad/SIQ-EGFP particles (100 μL), as indicated. Mice were treated with Teb by i.p. injection (100 μL, 1 mmol/L) once a day at 24-h intervals for 2 days. PBS alone was used as a control. EGFP expression in the liver was analyzed by immunostaining with an anti-EGFP antibody. (D) The durability of Ad/SIQ in the mouse liver. One day after injection of 100 μL Ad/SIQ-EGFP (107 virus particles/μL), Teb was administered twice (100 μL, 1 mmol/L) at a 24-h interval via i.p. injection. Mice were sacrificed at the indicated time points for the analysis of the expressivity of the EGFP transgene in the liver. Constitutively active Ad/EGFP under the control of the CMV promoter was used as a positive control. For the negative controls, indicated combinations of Ad/SIQ-EGFP and mock vehicles were delivered into the mice through the same procedures as the experimental group, but their livers were removed at day 3 only for immunostaining against EGFP. i.p., intraperitoneal injection; i.v., intravenous injection. Scale bar, 100 μm.

Figure 6

Accelerated reversibility of SIQmate (A) Schematic diagram of the SIQmate system. CymR and IQ-Driver are constitutively expressed by the CMV promoter, linked by a viral P2A peptide. CuO is strategically positioned directly upstream of a transcriptional start site. CymR binds to the CuO element, suppressing transgene expression by physically hindering RNA polymerase progression. Cumate interferes with CymR binding to CuO, enabling transgene induction. Therefore, the SIQmate switch requires two orthogonal chemical inducers, Teb and cumate, for transgene activation. (B) Transient transfection of HEK293 cells with pSIQmate-EGFP. After 24 h of incubation, the transfectants were treated with the indicated chemical inducers for an additional 24 h. Fluorescence was observed under the microscope. Scale bar, 200 μm. (C) Responsiveness of an established stable cell line with SIQmate-EGFP to concomitant treatment of Teb and cumate as indicated. Prolonged incubation of the cells with both chemical stimuli increased the strength of EGFP signals. Scale bar, 200 μm. (D) FACS analysis of the established SIQmate-EGFP cell line after chemical stimulation. (Left) Segregation of cells after chemical treatment, as indicated, based on the strength of EGFP fluorescence. Quantitative analysis of the yield of EGFP-positive cells is presented in the right panel. Cells were treated with 10 μM Teb and 1× cumate for 24 h before sorting the EGFP-positive cells (n = 3). (E) Direct comparison of the rapidity of transcript disappearance between SIQ and SIQmate systems after removing both Teb and cumate. Established cell lines of SIQ-EGFP and SIQmate-EGFP integrated at the attP locus of HEK293 cells were treated with both chemicals for 24 h. After the removal of the chemical stimulators by thorough washing, the EGFP transcript levels were measured by RT-qPCR at the indicated time points. Notably, the reduction in EGFP mRNA was more prominent under the control of SIQmate than that of SIQ. Statistical significance was addressed using two-way ANOVA with Šidák correction (n = 3). Scale bar, 200 μm. The data are presented as mean ± SD. ∗∗∗p < 0.001.