Plasmid construction

All promoter, gene and polyA signal were obtained via polymerase chain reaction (PCR) using the primers listed in S1 Table. The Tet-On 3G gene and CMV promoter (Pcmv) were cloned from a pCMV-Tet3G vector (Clontech) and the TRE3G promoter and firefly luciferase gene were cloned from a pTRE 3G-Luc vector (Clontech). The csy4 gene was amplified from a pSQT1601 plasmid, which was a generous gift from Keith Joung (Addgene plasmid # 53369). The tTS gene was cloned from pTet-tTS plasmid (Clontech). The mcherry and the kid gene (GenBank accession no. NC_022885.1) were cloned from pmcherry-kid plasmid. The White spot syndrome virus immediate-early 1 promoter (Pwssvie1) and the 23-bp truncated WSSV IE1 promoter (Pwssvie1 (Δ23)) were amplified from White spot syndrome virus (GenBank accession no. AF440570). The Orgyia pseudotsugata nucleopolyhedrosis virus (OpMNPV) immediate-early 2 promoter (Popie2), OpMNPV IE2 polyadenylation signal (opie2pA) and OpMNPV major envelope glycoprotein (GP64) polyadenylation signal (opgp64pA) were cloned from OpMNPV (GenBank accession no. NC_001875.2). The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early 1 (IE1) promoter (Pacie1) was cloned from AcMNPV (GenBank accession no. NC_001623). The Simian vacuolating virus 40 polyadenylation signal (sv40pA) and the Herpesvirus thymidine kinase polyadenylation signal (HSVTKpA) were cloned from pFastBac Dual plasmid (Invitrogen).

An mCherry expression cassette was constructed by inserting the WSSVIE1 promoter PCR fragment, mCherry gene, and opgp64 polyA signal into a pXL-BacII vector at BamHI–SpeI sites. The resultant plasmid was designated plasmid 1 (p1, Fig 1A). Plasmid 2 was obtained by inserting a Popie2-kid-opie2pA expression cassette into plasmid 1 at BamHI–XhoI sites (p2, Fig 1A), whereas plasmid 3 (p3, Fig 2A) was constructed by inserting a Popie2-Tet-On 3G-opie2pA expression cassette into plasmid 2 at BamHI–XhoI sites and a TRE3G-kid-sv40pA expression cassette at NsiI-XhoI sites. A Pacie1-tTS-HSVTKpA expression cassette was inserted into plasmid 3 at EcoRI–SpeI sites to produce plasmid 4 (p4, Fig 2A), and plasmid 5 (Fig 3A) was obtained by inserting the firefly luciferase gene into plasmid 3 to replace the kid gene at NotI–SalI sites. Plasmid 6 (Fig 3A) was obtained by replacing the kid gene with luciferase gene at NotI–SalI sites in plasmid 4. Plasmid 7 was obtained by inserting a csy4 recognition site between the TRE3G promoter and the luciferase gene by replacing the TRE3G promoter with TRE3G promoter and csy4 recognition site at NsiI–NotI sites in plasmid 6, and insertion of the Pcmv-csy4-HSVTKpA expression cassette into this plasmid (Fig 3A). Replacement of the cmv promoter in plasmid 7 with a wssvie1 (Δ23) promoter produced plasmid 8 (p8, Fig 3A), whereas plasmid 9 was constructed by replacing the Pacie1-csy4-HSVTKpA expression cassette with a Pcmv-csy4-HSVTKpA expression cassette (p9, Fig 3A). Plasmid 10 (p10, Fig 3A) was generated by inserting a TRE3G-rcsy4 expression cassette at an EcoRI site in plasmid 8, whereas plasmid 11 (p11, Fig 4A) was obtained by replacing the luciferase gene with kid gene at NotI–SalI sites in plasmid 10. All the sequences of the plasmids used in this study were listed in S1 File.

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Fig 1. Expression of Kid causes cell death in sf9 cells.

(A) A schematic diagram of vectors. Plasmid 1 (p1): the mcherry expression cassette was inserted into pXL-BacII vector. Plasmid 2 (p2): the kid expression cassette was added to plasmid 1. (B) Count of red fluorescence-positive cells in (C). Means and standard deviation were determined from three representative images (one from each of three independent biological replicates). (C) p1 and p2 were transfected into sf9 cells and the photographs were taken at 2 days post-transfection. The red-circled cells are fragmented and dead cells, illustrating the effects of Kid expression. Scale bar = 50 μm.

https://doi.org/10.1371/journal.pone.0244732.g001

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Fig 2. Leaky expression of the Tet-On 3G and Tet-On 3G-tTS systems.

(A) A schematic diagram of vectors. Plasmid 3 (p3): the kid gene expression was controlled by TRE3G promoter control, and the Tet-On 3G transcriptional activator protein was controlled by the Opie2 promoter. Plasmid 4 (p4): Added the tTS gene expression cassette to the p3 plasmid. (B) Count of red fluorescence-positive cells in (C). Means and standard deviation were calculated from three representative pictures (one from each of three independent biological replicates). Values followed by different letters with a column are significantly different at P < 0.05 according to one-way ANOVA. (C) Sf9 cells were transfected with plasmid p1, p2, p3, or p4, and 6 h later, doxycycline (50ng/μL) was added to the medium as indicated. The fluorescence expression and status of the insect cells were observed under a microscope at 48 h after transfection. The circled cells are fragmented and dead cells. Scale bar = 50 μm.

https://doi.org/10.1371/journal.pone.0244732.g002

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Fig 3. Reduction in the background expression level of the Tet-On 3G system using the csy4 system.

(A) A schematic diagram of vectors. Plasmid 5 (p5): control plasmid, firefly luciferase expression was controlled by the Tet-On 3G system. Plasmid 6 (p6): contained an additional tTS expression cassette compared to plasmid 5. Plasmid 7 (p7): Added a 28 bp csy4 recognition sequence between the TRE3G promoter and the luciferase gene, and added a csy4 gene which was driven by CMV promoter. Plasmid 8 (p8) and plasmid 9 (p9) were similar to plasmid 7 expect that CMV promoter was replaced with WSSVie1(Δ23) or Acie1 promoter. Plasmid 10 (p10): Compared to plasmid 8, there was an additional reverse csy4 expression cassette controlled by TRE3G promoter. (B) The csy4 28-nucleotide substrate sequence and the fragment after digestion are shown above. The black triangle indicates the cleavage site. (C) RT-qPCR analysis for the change of firefly luciferase transcript at 48 h post-transfection with the indicated plasmids. The luciferase relative transcript level of plasmid 5-transfected cells in the absence of doxycycline was set at 100%. (D) Relative luciferase activity was measured and calculated at 48 h post-transfection with the indicated plasmids. The relative luciferase activity values of plasmid 5-transfected cells in the absence of doxycycline was set at 100%. The background expression level and fold induction are indicated above each column without and with doxycycline induction, respectively. Error bars indicate the standard deviation calculated from three independent experiments.

https://doi.org/10.1371/journal.pone.0244732.g003

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Fig 4. The Tet-On 3G-Csy4 system tightly regulates kid gene expression.

(A) Vector construction diagram. Plasmid 11 (p11): The luciferase gene in plasmid p10 was replaced with the kid gene sequence. (B) Count of red fluorescence-positive cells in (C). Means and standard deviation were calculated from three representative images (one from each of three independent biological replicates). Values followed by different letters with a column are significantly different at P < 0.05 according to one-way ANOVA. (C) The fluorescence expression and cell growth were observed in sf9 cells at 48 h after transfection with p3, p4, or p11 in the absence or presence of doxycycline (50ng/μL). The circled cells are fragmented and dead cells. Scale bar = 50 μm.

https://doi.org/10.1371/journal.pone.0244732.g004

Cell culture and transfection of the sf9 cell line

sf9 cells were maintained at 27°C in TNM-FH medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (GIBCO). Plasmids for transfection were purified using a Plasmid Mini Kit (Omega), and concentrations were determined by using a NanoDrop 2000 spectrophotometer (Thermo). sf9 cells were seeded in 24-well plates and transfected with 1 μg plasmid using Cellfectin II reagent (Invitrogen) according to the manufacturer’s protocol. All transfections were performed independently three times in triplicate.

Fluorescence microscopy

sf9 cells were transfected with different plasmid as described in the previous section and were imaged at 48 h post-transfection with an Olympus IX71 imaging system using an RFP light cube or bright field.

Luciferase reporter assays

In order to determine levels of Tet-On 3G system-associated background expression, we used a dual-luciferase system to normalize cell numbers and transfection efficiency. Cells were grown in the absence or presence of doxycycline (50 ng/mL) in 24-well plates. The vectors and internal reference plasmid pxl-BacII-Rluc, which can express Renilla luciferase in sf9 cells, were co-transfected into the cells using Cellfectin II reagent (Invitrogen) according to the manufacturer’s protocol. Relative luminescence was measured at 48 h post-transfection using a Dual Luciferase Reporter assay kit (Transgen). Luminometric analysis was conducted using a PerkinElmer infinite 200 plate reader (Tecan).

Real-Time quantitative Polymerase Chain Reaction (RT-qPCR)

RT-qPCR was performed to determine the transcript levels. Plasmid transfection was the same as luciferase analysis. Samples were collected 48 hours after transfection. The total RNA was isolated using Trizol regent (Takara) according to instructions of the manufacturer. The cDNA was synthesized from the total RNA using the cDNA Reverse Transcription Kit (Takara) in accordance with manufacturer’s instructions. SYBR Green qPCR Master Mix (Transgen), specific primers of target firefly luciferase gene and internal control renilla luciferase genes were used in PCR reactions. PCR conditions were initial denaturation at 95°C for 30s, followed by 45 cycles at 95°C for 10 s and 60°C for 30 s. The CT values of firefly luciferase gene were normalized to the CT value of renilla luciferase gene and the relative fold expression changes were estimated by 2-[Delta][Delta] CT method.

Statistical analysis

All experiments were performed in triplicate with three independent biological replicates, and the results were expressed as the mean ± standard deviation (SD). Data were analyzed using one-way ANOVA and Tuckey’s multiple comparison test, with differences between groups being considered significant at the p < 0.05 level. All statistical tests were conducted using SPSS 25 software.

The kid protein showed strong cytotoxicity against sf9 cells

The sf9 insect cell line is commonly used in biological experiments, and typically serves as an insect platform for the production of proteins of interest [20, 21], as well as viruses [22]. Previously, it has been reported that the Kid toxin can induce apoptosis in mammalian cells [14], and therefore we examined whether Kid is also toxic to insect cells. The plasmid that can express Kid and mCherry were constructed. The control plasmid can only express red fluorescent protein. At 48 h post-transfection, expression of red fluorescence in cells transfected with kid gene was barely detectable when observed under a fluorescence microscope, with only cell fragments being mainly observed. However, red fluorescence in cells transfected with control plasmid could be clearly observed after transfection into sf9 cells (Fig 1B and 1C). These observations thereby indicate that the Kid toxin is highly cytotoxic to sf9 cells.

The presence of tTS did not reduce the background expression level of the Tet-On 3G system in sf9 cells

In the present study, we used the Tet-On 3G system to control expression of the Kid toxin in sf9 cells; however, cell death was still observed even in the absence of doxycycline induction (Fig 2B and 2C, p3), thereby indicating that the background expression level is still too high when this system is used to express highly toxic proteins.

In mammalian cells, the transcription silencing suppressor (tTS) is commonly used to reduce background expression of the Tet-on system, and thus in the present study, we introduced tTS to reduce the leaky expression of Kid driven by the Tet-On system. Nevertheless, following transfection into sf9 cells, we still observed high cell death rates, even in the absence of doxycycline induction (Fig 2B and 2C, p4), and we detected no significant effect of tTS incorporation with respect to controlling the background expression of Kid and levels of cell death (comparing p4 with p3 in Fig 2B). We accordingly deduced that tTS does not achieve the desired effect of controlling background expression in sf9 cells, and thus sought to identify other strategies that could be deployed to reduce the background expression level of the Tet-On 3G system.

Reducing the background expression level of the Tet-On 3G system using the Csy4 system

Given that, in contrast to mammalian cell, the tTS was ineffective in controlling background expression levels of the Tet-On 3G system in sf9 cells, we needed to develop alternative strategies to reduce background expression. Previous approaches used to regulate gene expression have primarily focused on transcriptional-level regulation. However, we speculated that post-transcriptional regulation, such as mRNA stability management, might be a promising approach to reduce the background expression level of the Tet-On 3G system. Accordingly, in the present study, we investigated the utility of the sequence-specific endoribonuclease Csy4 as a mediator of post-transcriptional regulation. To this end, we inserted the recognition sequence of Csy4 in the 5’ UTR region of the target gene, and used an alternative promoter to control the expression of Csy4. On the basis of previous studies, we had established that excessive expression of Csy4 is toxic to sf9 cells, and thus in order to identify suitable promoters for Csy4 expression, we constructed plasmids containing different promoters for controlling csy4 expression and conducted luciferase activity analyses.

For this purpose, we selected Acie1, WSSVie1 (Δ23) [23], and CMV promoters to control the expression of Csy4 due to their lower activities in insect cells. As shown in Fig 3, the original Tet-On 3G system had a high background expression level, with relative luciferase activities of 100% and 5171 ± 929.8% without and with doxycycline induction, respectively, representing a 51.7-fold induction effect. When tTS was incorporated into the system to control the leaky expression, the relative transcript level reduced to 60.4% and the relative luciferase activity reduced to 56.6% in the absence of doxycycline induction. However, for the expression of highly toxic proteins, this level of background expression is still too high to be acceptable. By replacing the tTS with Csy4, we observed that the background expression level of the target gene was reduced, and in response to an increase in promoter activity in sf9 cells for Csy4 expression, the leaky expression of the target gene was also reduced, for CMV, wssvie1 (Δ23) and acie1 promoters, the relative transcript levels were 21.56 ± 5.65%、3.71 ± 0.88%、1.76 ± 0.10%, the relative luciferase activities were 19.32 ± 2.06%, 0.71 ± 0.18%, 0.45 ± 0.20%, respectively. Nevertheless, the maximum expression level after doxycycline induction also decreased correspondingly. Accordingly, we found that all three of the promoters we assessed for driving Csy4 expression were either too weak to decrease the background expression of the target gene (CMV), or too strong, such that excess Csy4 expression markedly affected post-induction expression of the target gene [WSSVie1 (Δ23), and Acie1]. Although at this point, we could have continued to screen for suitable promoters for Csy4 expression, we considered that such a strategy would have taken too much time, and thus we adopted an alternative approach to overcome this problem.

The paradox regarding the use of Csy4 is that a certain level of this enzyme is required before dox-induction to reduce leaky expression, whereas the absence of Csy4 is required for maximal expression upon dox-induction. Accordingly, to remove needless Csy4 following the addition of doxycycline, we inserted a TRE3G promoter-driven reverse csy4 expression cassette into p8 (Fig 3A) to obtain p10. Our rationale in this regard is that high levels of anti-sense csy4 mRNA produced via the TRE3G-reverse csy4 cassette after doxycycline addition would form dsRNA with sense csy4 mRNA produced by the WSSVie1 (Δ23)-Csy4 cassette, and therefore knock down Csy4 expression [24], and the anti-sense csy4 was not toxic to sf9 insect cells (data not shown), such that the induction of target gene expression would not be affected by Csy4-mediated digestion.

We found that although the production of anti-sense csy4 mRNA resulted in a moderate increase in target gene background expression, as indicated by a change in relative luciferase activity from 0.71 ± 0.18% to 3.80 ± 0.78%, there was a considerably more pronounced increase in the induced expression of the target gene, with the relative luciferase activity changing from 17.80 ± 2.62% to 1541.80 ± 245.79%, and with a corresponding fold induction increase from 25.0 to 406.0 (Fig 3D). Compared with the induction obtained using p5 and p6, the maximum induced expression decreased to 30% and 18%, respectively; however, the fold increase in induction obtained using p10 was significantly higher than that obtained with either p5 or p6. The 406.0-fold increase in expression thus obtained would satisfy the requirements of most experiments, and the observed reduction in maximum expression could be compensated by transfecting with larger amounts of plasmid.

The Tet-On 3G-Csy4 system tightly regulated toxin expression

As noted previously, when using either the Tet-On 3G, or Tet-On 3G-tTS system, high levels of leaky Kid expression caused significant cell death (Fig 2B and 2C), indicating an insufficient control of target gene expression in both systems. However, in the system characterized by WSSVie1 (Δ23)-driven csy4 and TRE3G-driven anti-sense csy4, the background expression of kid is tightly controlled, without impairing the induction of expression, as little cell death was observed in the absence of doxycycline induction, whereas substantial cell death was detected in the presence of doxycycline (Fig 4B and 4C). These observations thus indicate that our modified Tet-On 3G-Csy4 system is more suitable for driving the inducible expression of toxin genes.