Development of all-in-one multicistronic Tet-On lentiviral vectors for inducible co-expression of two transgenes
∗
Yide Huang1,2 Ruonan Zhen1 Meiqin Jiang1 Jie Yang1 Yun Yang1 Zhen Huang1 Yao Lin1,2
1 College
of Life Sciences, Fujian Normal University, Fuzhou, Fujian, People’s Republic of China
2 Fujian
Key Laboratory of Developmental and Neural Biology, Fuzhou, Fujian, People’s Republic of China
Abstract Inducible co-expression of multiple genes is often needed in research. Here we describe a single-vector-based Tet-On inducible system for co-expression of two transgenes. The two transgenes (DsRed1 and eGFP as model genes) and reverse tetracycline-controlled transactivator were separated by internal ribosomal entry sites and 2A sequences, and their transcription was controlled by the same tetracycline responsive element. Two novel vectors with different internal
ribosomal entry sites and 2A positions on the vectors were constructed. The DsRed1 and eGFP in cells transduced with both vectors are undetectable in the absence of doxycycline and can be efficiently induced in the presence of doxycycline in vitro and in vivo. These two vectors can be useful tools when regulated co-expression of two ecotopic genes is C 2014 International Union of Biochemistry and Molecular needed. Biology, Inc. Volume 00, Number 0, Pages 1–7, 2014
Keywords: multiple-genes expression, Tet-On inducible system, doxycycline, lentiviral vector
1. Introduction Lentivirus, a subgroup of retroviruses, has the ability to integrate into the genome of both dividing and nondividing cells and offers stable transgene expression [1–3]. It is considered one of the most promising vehicles to efficiently deliver transgenes into cells. However, transgenes delivered by Lentivirus are often driven by constitutive promoters and do not really reflect the physiological gene expression level. Therefore, inducible systems were invented for more fine-tuned ectopic gene expression. Among all inducible systems, the best charac-
Abbreviations: rtTA, reverse tetracycline-controlled transactivator; TRE, tetracycline responsive element; IRES, internal ribosomal entry sites; FMDV, foot-and-mouth disease virus; EMCV, encephalomyocarditis virus. ∗ Address for correspondence: Yide Huang, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350108, People’s Republic of China; Tel.: 0086-591-22860592; Fax: 0086-591-22860592; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 14 January 2014; accepted 6 May 2014
DOI: 10.1002/bab.1239 Published online in Wiley Online Library (wileyonlinelibrary.com)
terized and most commonly used is the tetracycline-inducible (Tet-inducible) system, which is divided into Tet-Off and Tet-On systems [4, 5]. In the presence of doxycycline (a tetracycline analog), transgene expression is suppressed in the Tet-Off system and induced in the Tet-On system [6, 7]. Because the efficiency of transgene induction is higher and there is no need for persistent administration of doxycycline to suppress gene expression in it, the Tet-On system is more widely used when compared with the Tet-Off system [7, 8]. The conventional Tet-On system includes two separate constructs: One contains the regulatory unit for the constitutive expression of reverse tetracycline-controlled transactivator (rtTA), and the other contains the tetracycline responsive element (TRE) for transgene induction [6, 9–11]. In the dual-vector Tet-On system, selection and screening are required to obtain cells transduced with both constructs for transgene expression, which is not only time-consuming and inefficient but also impossible in vivo. To overcome this problem, single lentiviral vectors containing all the elements required for Tet-On system have been developed by several groups [12–16]. Simultaneous expression of multiple genes is often needed in research. It will be useful to develop a Tet-On system that would allow multiple-transgene induction. Many strategies such as fusion proteins, multiple promoters, mRNA splicing, and cleavage factors can be employed to express multiple genes.
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Biotechnology and Applied Biochemistry A common strategy to evenly express two transgenes from one construct is the insertion of an internal ribosomal entry sites (IRES) element or a 2A sequence between the two transgenes under the control of single promoter [17–19]. IRES elements are able to attract the eukaryotic ribosome to the mRNA molecule and, therefore, allow internal initiation of translation in a capindependent manner [20]. The 2A sequence encodes a highly conserved consensus motif -DVExNPGP-. During translation, the polypeptide breaks between the glycine and the proline at the end of this motif, but translation of the sequence after this motif continues until a stop codon is met. When the 2A sequence is used for two-transgene expression, the C-terminus of the upstream protein is fused with the 2A peptide (except the last proline), and the downstream protein is released intact (with the addition of an N-terminal proline) [17]. In this article, we describe a strategy for co-expression of two proteins, using a single-vector-based Tet-On inducible system. Enhanced green fluorescent protein (eGFP) and red fluorescent protein (DsRed1) were employed as model genes. The efficacy of the 2A sequence from the foot-and-mouth disease virus (FMDV) and IRES from the encephalomyocarditis virus (EMCV) in separating eGFP, Dsred, and rtTA and initiating translation were compared in vitro and in vivo. Our results demonstrated that the Tet-On vectors we developed were efficient in evenly inducing two-transgene expression and will be useful and simple tools for biological research.
sites at both ends was amplified from the pDsRed1–N1 vector (F-primer: 5 -TGGGCGTGGATAGCGGTTTGACTCA-3 ; R-primer: 5 -CCGACGTCGACCAGAACAGGTGG-3 ) and cloned into the TREAutoR3/2A–eGFP–IRES–rtTA to create the DsRed1–2A– eGFP–IRES–rtTA3 vector. For the DsRed1–IRES–eGFP–2A–rtTA3 vector, the IRES–eGFP fragment with 5 SalI and 3 XhoI sites was first obtained from the pIRES2–eGFP plasmid (Fprimer: 5 -GCTCAAGCTTCGAATTCTGCAGTC-3 ; R-primer: 5 -TACCGCTCGAGCTTGTACAGCTCGT-3 ) and cloned into pBluescript/2A to create pBluescript/IRES–eGFP-2A. Then, the DsRed1 fragment (including stop codon) with SalI sites at both ends was amplified from the pDsRed1–N1 vector (F-primer: 5 -AGCGCTACCGGACTCAGATCTCG-3 ; R-primer: 5 -CCGACGTCGACATTATGATCTAGAGTC-3 ) and cloned into pBluescript/IRES–eGFP–2A to create pBluescript/DsRed1– IRES–eGFP–2A. To remove the eGFP–IRES fragment from TREAutoR3 and to clone DsRed1–IRES–eGFP–2A into TREAutoR3, an EcoRI site between IRES and rtTA3 was created by overlap extension PCR. The DsRed1–IRES–eGFP– 2A fragment flanked by EcoRI at the 5 and 3 ends was amplified from pBluescript/DsRed1–IRES–eGFP–2A (Fprimer: 5 -CCGGAATTCACCGGTGATATCGTCG-3 ; R-rimer: 5 GACACTCCGGATTTTCCCAGTCACG-3 ) and cloned into TREAutoR3 to create the DsRed1–IRES–eGFP–2A–rtTA3 construct.
2.3. Lentiviral vector preparation and titration
2. Materials and Methods 2.1. Ethics statement This study was carried out in accordance with the Chinese Regulation for the Administration of Affairs Concerning Experimental Animals. The protocol was permitted by the Ethics Committee of Fujian Normal University. The mouse was killed by the “breaking the neck” protocol, and all efforts were made to minimize suffering.
2.2. Plasmid construction TREAutoR3 [12] was a kind gift from Dr. J. Seppen (AMC Liver Center of the Academic Medical Center at the University of Amsterdam) and used as a template to construct the tricistronic Tet-On lentiviral vector via a series of subcloning steps. For DsRed1–2A–eGFP–IRES–rtTA3 vector, first, the 2A fragment from the FMDV flanked by AgeI and SalI at the 5 end and PstI at the 3 end was synthesized by TaKaRa Lt Co. (Dalian, China) and cloned into the NotI and PstI sites of pBluescript II KS (+) vector to produce pBluescript/2A. Second, the eGFP fragment with the 5 EcoRI site and 3 PstI site was amplified from the pEGFPN1 vector (F-primer: 5 -CCGGAATTCATGGTGAGCAAGG-3 ; Rprimer: 5 -GCGCCCTGCAGTTACTTGTACAGC-3 ) and cloned into the pBluescript/2A construct to create pBluescript/2A–eGFP vector. Third, the 2A–eGFP fragment from pBluescript/2A– eGFP was cloned into the TREAutoR3 by using the AgeI and PstI sites to create the TREAutoR3/2A–eGFP–IRES–rtTA vector. Fourth, the DsRed1 fragment (without stop codon) with SalI
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The lentiviral vector particles were produced by three-plasmid transient transfection into 293T cells as described earlier [21]. Briefly, 293T cells plated at 70% confluence were cotransfected with envelope plasmids (pVSVG), packaging plasmids (pHelper), and the appropriate gene transfer vector by using calcium phosphate precipitation. Twelve hours later, fresh Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; HyClone, CA, USA) was added. The viral particles were collected 24 H later and fresh medium was added to the cells. Forty-eight hours later, the viral particles were collected again. Then the viral particles were concentrated by centrifugation at 50,000g, 4 ◦ C for 2.5 H. The pellet viral particles were resuspended in fresh DMEM with 10% FBS and stored at −80 ◦ C. A serial dilution method was used to calculate the virus titer. Briefly, the concentrated virus stock was diluted and transfected into 293T cells. Twelve hours later, the cells were induced with 1 μg/mL doxycycline for 72 H, and the expression of fluorescent proteins was examined to calculate the virus titer.
2.4. Cell culture and treatment of doxycycline 293T (human embryonic kidney) cells were cultured at 37 ◦ C with 5% CO2 in standard DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Doxycycline (Sigma-Aldrich, MO, USA) was diluted in water and filtersterilized to make 10 mg/mL stock stored at −20◦ C. In typical experiments, 12 H after virus infection, the culture media were replaced with fresh media containing respective concentrations
All-in-One Multicistronic Tet-On Lentiviral Vectors
FIG. 1
Schematic diagram of the constructed tricistronic Tet-On lentiviral vectors. The bicistronic vector TREAutoR3 [12] was used to create tricistronic vectors containing DsRed1, eGFP, and rtTA3 genes sequentially. Depending on the order of the 2A and IRES sequences, two tricistronic vectors, (A) DsRed1–2A–eGFP–IRES–rtTA3 and (B) DsRed1–IRES–eGFP–2A–rtTA3, were constructed. On both vectors, transcription of all three genes is controlled by TRE containing a minimal cytomegalovirus promoter fused to seven copies of TetO.
of doxycycline, and the cells were left to grow for the indicated length of time before the harvest.
2.5. Western blotting After the harvest, cells were lysed in urea lysis buffer (20 mM HEPES, pH 8.0; 6.25 M urea; 25 mM NaCl; 100 mM dithiothreitol; 0.05% Triton 100). Cell lysate (30 μg) was separated on a 12% SDS-PAGE. Protein bands were transferred to HybondC nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK) through electroblotting. The membranes were blocked with 5% fat-free milk and probed overnight at 4 ◦ C with primary antibodies, specifically against eGFP, DsRed, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alexa Fluor 488 donkey anti-mouse immunoglobulin G (Invitrogen, San Diego, CA, USA) was used as the secondary antibody at 1:1,000 dilutions. Detection and quantification of proteins were performed using Fluor Chem Q (Cell Biosciences, Santa Clara, CA, USA) and Scion Image software.
2.6. In vivo induction of the lentiviral vectors For in vivo induction, A375 (human malignant melanoma) cells were cultured at 37 ◦ C with 5% CO2 in standard DMEM supplemented with 10% FBS. When the cell confluence reached about 70%, A375 cells were infected with the respective virus vectors and left to grow for 12 H before the media were replaced with fresh DMEM. Forty-eight hours later, cells were trypsinized and injected subcutaneously to the left-flank regions of nude mice to generate tumors. Before excision, for 3 weeks, half the mice were fed with water containing doxycycline (200 μg/mL), and the other half were fed with only water as negative control. The tumors were fixed overnight with 4% paraformaldehyde in phosphate-buffered saline (PBS). Frozen sections were cut
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from tumors, rinsed twice in PBS, transferred to 30% sucrose in PBS for 5 H, and then frozen in optical coherence tomography mounting medium. Twelve-micrometer-thick sections were obtained with a cryostat (Microm, Walldorf, Germany) and analyzed using a confocal microscope (Nikon, Tokyo, Japan).
3. Results 3.1. Generation of tricistronic Tet-On lentiviral vectors To generate the tricistronic Tet-On lentiviral vectors, we employed the TREAutoR3 vector generated by Seppen and coworkers [12] as a template. eGFP and DsRed1 were chosen as model genes to validate the expression efficiency of the new vectors. Therefore, the target vectors would include three genes, namely, eGFP, DsRed1, and rtTA3 (rtTAS12G F86Y A209T ), the transcription of which were all controlled by the TRE element (Fig. 1). Two tricistronic vectors were designed: (1) DsRed1–2A– eGFP–IRES–rtTA. In this construct, the 2A sequence was placed between Dsred1 and eGFP, and the IRES sequence separated eGFP and rtTA3 (Fig. 1A). Moreover, the DsRed1 gene lost its stop codon and was fused in frame with 2A and eGFP. (2) DsRed1–IRES–eGFP–2A–rtTA, in which the position of 2A and IRES sequences in the construct DsRed1–2A–eGFP–IRES–rtTA mentioned above was swapped (Fig. 1B). In addition, the eGFP gene lost its stop codon and was fused in frame with 2A and rtTA3 in this construct.
3.2. Rapid and efficient induction of the tricistronic vectors by doxycycline After the vector construction, we evaluated the efficiency of doxycycline induction in cells transduced with the tricistronic vectors. First, we investigated how quickly the transgenes can be induced by performing a time course of excessive doxycycline (1 μg/mL in the media) treatment (0, 8, 24, 48, and 72 H) in 293T cells transduced with the two vectors. The cells infected with the DsRed1–2A–eGFP–IRES–rtTA vector displayed a much more rapid induction in comparison with those infected with the DsRed1–IRES–eGFP–2A–rtTA vector (peak time: 24 H vs. 48 H) (Fig. 2). Next, we investigated the vector sensitivity toward doxycycline, using a titration of doxycycline (0, 50, 100, 200, 500, and 1000 ng/mL in the media) in transduced 293T cells. Both vectors were very sensitive to doxycycline and can be strongly induced at low concentrations (Fig. 3). However, the induction of DsRed1–2A–eGFP–IRES–rtTA is slightly higher than
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FIG. 2
Time-dependent induction of DsRed1 and eGFP in response to doxycycline treatment in 293T cells transduced with the tricistronic vectors. 293T cells transduced with (A) DsRed1–2A–eGFP–IRES–rtTA3 or (B) DsRed1–IRES–eGFP–2A–rtTA3 were treated with doxycycline (1 μg/mL in the media) and harvested at 0, 8, 24, 48, and 72 H posttreatment. Expression of DsRed1 and eGFP proteins were analyzed using Western blot. Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panels of both (A) and (B).
that of DsRed1–IRES–eGFP–2A–rtTA3 at 50 ng/mL (Fig. 3A and 3B), suggesting a more efficient induction of the former vector. A big advantage of the template TREAutoR3 vector over other Tet-On systems is the low basal expression of the transgene [12]. Therefore, we evaluated the basal expression of eGFP and DsRed1 in 293T cells transduced with DsRed1–2A–eGFP– IRES–rtTA, DsRed1–IRES–eGFP–2A–rtTA, or TREAutoR3, using fluorescence microscope and Western blot. Similar to cells infected with TREAutoR3, the basal expression of both eGFP and DsRed1 in cells infected with our vectors is very low (Fig. 4). When these transduced 293T cells were exposed to media containing 1 μg/mL doxycycline for 48 H, the induction of the transgenes on all three vectors were similar too, suggesting our vectors did not alter the low basal expression and high efficiency of the template TREAutoR3 vector.
3.3. Induction of the tricistronic vectors in vivo Next we set out to investigate whether the tricistronic vectors we created can be induced in vivo, using a mouse xenograft model of A375 malignant melanoma cell line. A375 cells were transduced with either DsRed1–2A–eGFP–IRES–rtTA3 or DsRed1–IRES–eGFP–2A–rtTA3 vectors at a multiplicity of infection of 60 and injected subcutaneously into the left-flank regions of nude mice to generate tumors. These mice were then fed with water with or without doxycycline (200 μg/mL)
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for 3 weeks before the tumors were excised. The expression of both eGFP and DsRed1 were clearly induced by doxycycline in the A375 xenograft transduced with both vectors (Fig. 5), suggesting these two vectors can be efficiently induced in vivo.
4. Discussion The IRES and 2A sequences are both widely reported nucleotide elements that are capable of initiating translation in the middle of mRNA. However, different from the IRES sequence, the 2A sequence cannot recruit ribosome on its own, and its function relies on the translation machinery started by the transgene before it. Therefore, when the 2A sequence is used to separate genes, the stop codon of the gene before the 2A sequence has to be removed. In this case, the peptide encoded by the 2A sequence (except the last proline) will be added to the end of the upstream transgene, and an extra proline will be added to the beginning of the downstream gene [17, 18]. Moreover, it has also been published that the separation effect of the 2A sequence is not complete, and it would allow the production of around 20% fusion proteins [22]. In the current work, a weak fusion band of DsRed1 and eGFP can also be observed when the 2A sequence is placed between them (Fig. S1), supporting the existence of fusion proteins created by the 2A sequence. Although the fusion proteins created by the 2A sequence contain the full sequence of both exogenous proteins, they may not maintain the functions of these proteins. For example, compared with DsRed1–2A–eGFP–IRES–rtTA, the sensitivity to and the induction speed of DsRed1–IRES–eGFP–2A–rtTA by doxycycline is slightly weaker (Figs. 2 and 3). This is probably due to the 2A-sequence-mediated production of dysfunctional eGFP–rtTA3 fusion proteins that can no longer cooperate with doxycycline or activate RNA polymerase to initiate transcription. More functional rtTA3 proteins in cells transduced with DsRed1–2A–eGFP–IRES–rtTA led to higher doxycycline sensitivity and faster induction. Therefore, IRES may be more suitable to be placed before rtTA3 for efficient
All-in-One Multicistronic Tet-On Lentiviral Vectors
FIG. 3
FIG. 4
Induction of DsRed1 and eGFP in response to a titration of doxycycline treatment in 293T cells transduced with the tricistronic vectors. 293T cells transduced with (A) DsRed1–2A–eGFP–IRES–rtTA3 or (B) DsRed1–IRES–eGFP–2A–rtTA3 were treated with the indicated concentrations of doxycycline (0, 50, 100, 200, 500, and 1000 ng/mL in the media) for 48 H before the harvest. Expression of DsRed1 and eGFP proteins was analyzed using Western blot. Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panels of both (A) and (B).
Comparison of the TREAutoR3 template vector with the tricistronic vectors. 293T cells transduced with the indicated vectors were treated with or without doxycycline (1 μg/mL in the media) for 48 H and subjected to (A) fluorescence microscope or (B) Western blot examinations for the expression of DsRed1 and eGFP proteins. (A) Scale bars = 100 μm. (B) Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panel.
doxycycline induction. However, in the DsRed1–2A–eGFP– IRES–rtTA transduced cells, both DsRed1 and eGFP proteins contained extra amino acids and fusion proteins were produced (Fig. S1). Therefore, DsRed1–2A–eGFP–IRES–rtTA is probably not suitable for the expression of proteins such as mouse double minute 2 homolog in which both the C-terminus and
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the N-terminus have important roles in regulating protein modification and interaction, but may be better for proteins such as death-associated protein kinase 1 whose terminal regions are not reported for critical regulatory roles [23]. An important problem for inducible system is leakiness, which is due to the unexpected translation of rtTA3 in
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FIG. 5
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Induction of DsRed1 and eGFP in response to doxycycline treatment in mouse xenografts transduced with the tricistronic vectors. A375 cells were transduced with either DsRed1–2A–eGFP–IRES–rtTA3 or DsRed1–IRES–eGFP–2A–rtTA3. Cells were implanted subcutaneously into the left-flank regions of nude mice (n = 8). The mice were then fed with water with or without doxycycline (200 μg/mL) continuously for 3 weeks before tumor excision. DsRed1 and eGFP induction were detected on frozen tumor sections through fluorescence confocal microscope. Scale bar = 100 μm.
All-in-One Multicistronic Tet-On Lentiviral Vectors
transduced cells when doxycycline is not present. Although both of our vectors have similar low basal expression compared with the template TREAutoR3 (Fig. 4), weak leakage of eGFP and DsRed1 proteins can still be observed in Western blot and fluorescence staining (Figs. 2–4). However, the expression of the transgenes from leakage was insignificant in comparison with when the transduced cells were exposed to doxycycline (Figs. 2–4). Therefore, unless the transgenes can affect cellular processes at very low level, our vectors should be suitable for most target genes in biological research. Nevertheless, it will be useful to have a better promoter other than TRE that would allow further reduction or even elimination of leakiness in the inducible system in the future. In addition, the sequence of the target genes may significantly affect the efficiency of translation [24]. In the current work, we only tested our vectors by using GFP and DsRed1 genes, which are not present in mammalian cells. However, when other “genes of interest” are applied in the future application of these vectors, there may be unexpected activation or silencing of the exogenous genes, caused by their endogenous regulators or codon usage bias. Recently, Stergachis et al. have shown that the coding region may also regulate the binding of transcription factors [25]. Therefore, more work will be needed to validate the further application of these vectors. In conclusion, we have created two all-in-one Tet-On tricistronic vectors expressing two trangenes under the control of a single TRE promoter. Similar to the template TREAutoR3 vector, both new vectors displayed low basal expression level and efficient doxycycline induction in vitro and in vivo, suggesting that they can be used as useful tools for inducible epigenetic gene expression in cells and animals. However, on the basis of the positions of the 2A and IRES sequences that are used to separate the two transgenes and rtTA3, each new vector has its advantages. Further application of these constructs may shed light on discovering more characteristics of the vectors and the translation machinery of the IRES and 2A sequences.
5. Acknowledgments This work was supported by the National Natural Science Foundation of China for Young Scholar (grant no. 31301172)
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and the Foundation of Fujian Educational Committee (grant no. JA12061). The authors have no conflict of interest.
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∗
Yide Huang1,2 Ruonan Zhen1 Meiqin Jiang1 Jie Yang1 Yun Yang1 Zhen Huang1 Yao Lin1,2
1 College
of Life Sciences, Fujian Normal University, Fuzhou, Fujian, People’s Republic of China
2 Fujian
Key Laboratory of Developmental and Neural Biology, Fuzhou, Fujian, People’s Republic of China
Abstract Inducible co-expression of multiple genes is often needed in research. Here we describe a single-vector-based Tet-On inducible system for co-expression of two transgenes. The two transgenes (DsRed1 and eGFP as model genes) and reverse tetracycline-controlled transactivator were separated by internal ribosomal entry sites and 2A sequences, and their transcription was controlled by the same tetracycline responsive element. Two novel vectors with different internal
ribosomal entry sites and 2A positions on the vectors were constructed. The DsRed1 and eGFP in cells transduced with both vectors are undetectable in the absence of doxycycline and can be efficiently induced in the presence of doxycycline in vitro and in vivo. These two vectors can be useful tools when regulated co-expression of two ecotopic genes is C 2014 International Union of Biochemistry and Molecular needed. Biology, Inc. Volume 00, Number 0, Pages 1–7, 2014
Keywords: multiple-genes expression, Tet-On inducible system, doxycycline, lentiviral vector
1. Introduction Lentivirus, a subgroup of retroviruses, has the ability to integrate into the genome of both dividing and nondividing cells and offers stable transgene expression [1–3]. It is considered one of the most promising vehicles to efficiently deliver transgenes into cells. However, transgenes delivered by Lentivirus are often driven by constitutive promoters and do not really reflect the physiological gene expression level. Therefore, inducible systems were invented for more fine-tuned ectopic gene expression. Among all inducible systems, the best charac-
Abbreviations: rtTA, reverse tetracycline-controlled transactivator; TRE, tetracycline responsive element; IRES, internal ribosomal entry sites; FMDV, foot-and-mouth disease virus; EMCV, encephalomyocarditis virus. ∗ Address for correspondence: Yide Huang, College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350108, People’s Republic of China; Tel.: 0086-591-22860592; Fax: 0086-591-22860592; e-mail: [email protected]. Supporting Information is available in the online issue at wileyonlinelibrary.com. Received 14 January 2014; accepted 6 May 2014
DOI: 10.1002/bab.1239 Published online in Wiley Online Library (wileyonlinelibrary.com)
terized and most commonly used is the tetracycline-inducible (Tet-inducible) system, which is divided into Tet-Off and Tet-On systems [4, 5]. In the presence of doxycycline (a tetracycline analog), transgene expression is suppressed in the Tet-Off system and induced in the Tet-On system [6, 7]. Because the efficiency of transgene induction is higher and there is no need for persistent administration of doxycycline to suppress gene expression in it, the Tet-On system is more widely used when compared with the Tet-Off system [7, 8]. The conventional Tet-On system includes two separate constructs: One contains the regulatory unit for the constitutive expression of reverse tetracycline-controlled transactivator (rtTA), and the other contains the tetracycline responsive element (TRE) for transgene induction [6, 9–11]. In the dual-vector Tet-On system, selection and screening are required to obtain cells transduced with both constructs for transgene expression, which is not only time-consuming and inefficient but also impossible in vivo. To overcome this problem, single lentiviral vectors containing all the elements required for Tet-On system have been developed by several groups [12–16]. Simultaneous expression of multiple genes is often needed in research. It will be useful to develop a Tet-On system that would allow multiple-transgene induction. Many strategies such as fusion proteins, multiple promoters, mRNA splicing, and cleavage factors can be employed to express multiple genes.
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Biotechnology and Applied Biochemistry A common strategy to evenly express two transgenes from one construct is the insertion of an internal ribosomal entry sites (IRES) element or a 2A sequence between the two transgenes under the control of single promoter [17–19]. IRES elements are able to attract the eukaryotic ribosome to the mRNA molecule and, therefore, allow internal initiation of translation in a capindependent manner [20]. The 2A sequence encodes a highly conserved consensus motif -DVExNPGP-. During translation, the polypeptide breaks between the glycine and the proline at the end of this motif, but translation of the sequence after this motif continues until a stop codon is met. When the 2A sequence is used for two-transgene expression, the C-terminus of the upstream protein is fused with the 2A peptide (except the last proline), and the downstream protein is released intact (with the addition of an N-terminal proline) [17]. In this article, we describe a strategy for co-expression of two proteins, using a single-vector-based Tet-On inducible system. Enhanced green fluorescent protein (eGFP) and red fluorescent protein (DsRed1) were employed as model genes. The efficacy of the 2A sequence from the foot-and-mouth disease virus (FMDV) and IRES from the encephalomyocarditis virus (EMCV) in separating eGFP, Dsred, and rtTA and initiating translation were compared in vitro and in vivo. Our results demonstrated that the Tet-On vectors we developed were efficient in evenly inducing two-transgene expression and will be useful and simple tools for biological research.
sites at both ends was amplified from the pDsRed1–N1 vector (F-primer: 5 -TGGGCGTGGATAGCGGTTTGACTCA-3 ; R-primer: 5 -CCGACGTCGACCAGAACAGGTGG-3 ) and cloned into the TREAutoR3/2A–eGFP–IRES–rtTA to create the DsRed1–2A– eGFP–IRES–rtTA3 vector. For the DsRed1–IRES–eGFP–2A–rtTA3 vector, the IRES–eGFP fragment with 5 SalI and 3 XhoI sites was first obtained from the pIRES2–eGFP plasmid (Fprimer: 5 -GCTCAAGCTTCGAATTCTGCAGTC-3 ; R-primer: 5 -TACCGCTCGAGCTTGTACAGCTCGT-3 ) and cloned into pBluescript/2A to create pBluescript/IRES–eGFP-2A. Then, the DsRed1 fragment (including stop codon) with SalI sites at both ends was amplified from the pDsRed1–N1 vector (F-primer: 5 -AGCGCTACCGGACTCAGATCTCG-3 ; R-primer: 5 -CCGACGTCGACATTATGATCTAGAGTC-3 ) and cloned into pBluescript/IRES–eGFP–2A to create pBluescript/DsRed1– IRES–eGFP–2A. To remove the eGFP–IRES fragment from TREAutoR3 and to clone DsRed1–IRES–eGFP–2A into TREAutoR3, an EcoRI site between IRES and rtTA3 was created by overlap extension PCR. The DsRed1–IRES–eGFP– 2A fragment flanked by EcoRI at the 5 and 3 ends was amplified from pBluescript/DsRed1–IRES–eGFP–2A (Fprimer: 5 -CCGGAATTCACCGGTGATATCGTCG-3 ; R-rimer: 5 GACACTCCGGATTTTCCCAGTCACG-3 ) and cloned into TREAutoR3 to create the DsRed1–IRES–eGFP–2A–rtTA3 construct.
2.3. Lentiviral vector preparation and titration
2. Materials and Methods 2.1. Ethics statement This study was carried out in accordance with the Chinese Regulation for the Administration of Affairs Concerning Experimental Animals. The protocol was permitted by the Ethics Committee of Fujian Normal University. The mouse was killed by the “breaking the neck” protocol, and all efforts were made to minimize suffering.
2.2. Plasmid construction TREAutoR3 [12] was a kind gift from Dr. J. Seppen (AMC Liver Center of the Academic Medical Center at the University of Amsterdam) and used as a template to construct the tricistronic Tet-On lentiviral vector via a series of subcloning steps. For DsRed1–2A–eGFP–IRES–rtTA3 vector, first, the 2A fragment from the FMDV flanked by AgeI and SalI at the 5 end and PstI at the 3 end was synthesized by TaKaRa Lt Co. (Dalian, China) and cloned into the NotI and PstI sites of pBluescript II KS (+) vector to produce pBluescript/2A. Second, the eGFP fragment with the 5 EcoRI site and 3 PstI site was amplified from the pEGFPN1 vector (F-primer: 5 -CCGGAATTCATGGTGAGCAAGG-3 ; Rprimer: 5 -GCGCCCTGCAGTTACTTGTACAGC-3 ) and cloned into the pBluescript/2A construct to create pBluescript/2A–eGFP vector. Third, the 2A–eGFP fragment from pBluescript/2A– eGFP was cloned into the TREAutoR3 by using the AgeI and PstI sites to create the TREAutoR3/2A–eGFP–IRES–rtTA vector. Fourth, the DsRed1 fragment (without stop codon) with SalI
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The lentiviral vector particles were produced by three-plasmid transient transfection into 293T cells as described earlier [21]. Briefly, 293T cells plated at 70% confluence were cotransfected with envelope plasmids (pVSVG), packaging plasmids (pHelper), and the appropriate gene transfer vector by using calcium phosphate precipitation. Twelve hours later, fresh Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; HyClone, CA, USA) was added. The viral particles were collected 24 H later and fresh medium was added to the cells. Forty-eight hours later, the viral particles were collected again. Then the viral particles were concentrated by centrifugation at 50,000g, 4 ◦ C for 2.5 H. The pellet viral particles were resuspended in fresh DMEM with 10% FBS and stored at −80 ◦ C. A serial dilution method was used to calculate the virus titer. Briefly, the concentrated virus stock was diluted and transfected into 293T cells. Twelve hours later, the cells were induced with 1 μg/mL doxycycline for 72 H, and the expression of fluorescent proteins was examined to calculate the virus titer.
2.4. Cell culture and treatment of doxycycline 293T (human embryonic kidney) cells were cultured at 37 ◦ C with 5% CO2 in standard DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Doxycycline (Sigma-Aldrich, MO, USA) was diluted in water and filtersterilized to make 10 mg/mL stock stored at −20◦ C. In typical experiments, 12 H after virus infection, the culture media were replaced with fresh media containing respective concentrations
All-in-One Multicistronic Tet-On Lentiviral Vectors
FIG. 1
Schematic diagram of the constructed tricistronic Tet-On lentiviral vectors. The bicistronic vector TREAutoR3 [12] was used to create tricistronic vectors containing DsRed1, eGFP, and rtTA3 genes sequentially. Depending on the order of the 2A and IRES sequences, two tricistronic vectors, (A) DsRed1–2A–eGFP–IRES–rtTA3 and (B) DsRed1–IRES–eGFP–2A–rtTA3, were constructed. On both vectors, transcription of all three genes is controlled by TRE containing a minimal cytomegalovirus promoter fused to seven copies of TetO.
of doxycycline, and the cells were left to grow for the indicated length of time before the harvest.
2.5. Western blotting After the harvest, cells were lysed in urea lysis buffer (20 mM HEPES, pH 8.0; 6.25 M urea; 25 mM NaCl; 100 mM dithiothreitol; 0.05% Triton 100). Cell lysate (30 μg) was separated on a 12% SDS-PAGE. Protein bands were transferred to HybondC nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK) through electroblotting. The membranes were blocked with 5% fat-free milk and probed overnight at 4 ◦ C with primary antibodies, specifically against eGFP, DsRed, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Alexa Fluor 488 donkey anti-mouse immunoglobulin G (Invitrogen, San Diego, CA, USA) was used as the secondary antibody at 1:1,000 dilutions. Detection and quantification of proteins were performed using Fluor Chem Q (Cell Biosciences, Santa Clara, CA, USA) and Scion Image software.
2.6. In vivo induction of the lentiviral vectors For in vivo induction, A375 (human malignant melanoma) cells were cultured at 37 ◦ C with 5% CO2 in standard DMEM supplemented with 10% FBS. When the cell confluence reached about 70%, A375 cells were infected with the respective virus vectors and left to grow for 12 H before the media were replaced with fresh DMEM. Forty-eight hours later, cells were trypsinized and injected subcutaneously to the left-flank regions of nude mice to generate tumors. Before excision, for 3 weeks, half the mice were fed with water containing doxycycline (200 μg/mL), and the other half were fed with only water as negative control. The tumors were fixed overnight with 4% paraformaldehyde in phosphate-buffered saline (PBS). Frozen sections were cut
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from tumors, rinsed twice in PBS, transferred to 30% sucrose in PBS for 5 H, and then frozen in optical coherence tomography mounting medium. Twelve-micrometer-thick sections were obtained with a cryostat (Microm, Walldorf, Germany) and analyzed using a confocal microscope (Nikon, Tokyo, Japan).
3. Results 3.1. Generation of tricistronic Tet-On lentiviral vectors To generate the tricistronic Tet-On lentiviral vectors, we employed the TREAutoR3 vector generated by Seppen and coworkers [12] as a template. eGFP and DsRed1 were chosen as model genes to validate the expression efficiency of the new vectors. Therefore, the target vectors would include three genes, namely, eGFP, DsRed1, and rtTA3 (rtTAS12G F86Y A209T ), the transcription of which were all controlled by the TRE element (Fig. 1). Two tricistronic vectors were designed: (1) DsRed1–2A– eGFP–IRES–rtTA. In this construct, the 2A sequence was placed between Dsred1 and eGFP, and the IRES sequence separated eGFP and rtTA3 (Fig. 1A). Moreover, the DsRed1 gene lost its stop codon and was fused in frame with 2A and eGFP. (2) DsRed1–IRES–eGFP–2A–rtTA, in which the position of 2A and IRES sequences in the construct DsRed1–2A–eGFP–IRES–rtTA mentioned above was swapped (Fig. 1B). In addition, the eGFP gene lost its stop codon and was fused in frame with 2A and rtTA3 in this construct.
3.2. Rapid and efficient induction of the tricistronic vectors by doxycycline After the vector construction, we evaluated the efficiency of doxycycline induction in cells transduced with the tricistronic vectors. First, we investigated how quickly the transgenes can be induced by performing a time course of excessive doxycycline (1 μg/mL in the media) treatment (0, 8, 24, 48, and 72 H) in 293T cells transduced with the two vectors. The cells infected with the DsRed1–2A–eGFP–IRES–rtTA vector displayed a much more rapid induction in comparison with those infected with the DsRed1–IRES–eGFP–2A–rtTA vector (peak time: 24 H vs. 48 H) (Fig. 2). Next, we investigated the vector sensitivity toward doxycycline, using a titration of doxycycline (0, 50, 100, 200, 500, and 1000 ng/mL in the media) in transduced 293T cells. Both vectors were very sensitive to doxycycline and can be strongly induced at low concentrations (Fig. 3). However, the induction of DsRed1–2A–eGFP–IRES–rtTA is slightly higher than
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FIG. 2
Time-dependent induction of DsRed1 and eGFP in response to doxycycline treatment in 293T cells transduced with the tricistronic vectors. 293T cells transduced with (A) DsRed1–2A–eGFP–IRES–rtTA3 or (B) DsRed1–IRES–eGFP–2A–rtTA3 were treated with doxycycline (1 μg/mL in the media) and harvested at 0, 8, 24, 48, and 72 H posttreatment. Expression of DsRed1 and eGFP proteins were analyzed using Western blot. Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panels of both (A) and (B).
that of DsRed1–IRES–eGFP–2A–rtTA3 at 50 ng/mL (Fig. 3A and 3B), suggesting a more efficient induction of the former vector. A big advantage of the template TREAutoR3 vector over other Tet-On systems is the low basal expression of the transgene [12]. Therefore, we evaluated the basal expression of eGFP and DsRed1 in 293T cells transduced with DsRed1–2A–eGFP– IRES–rtTA, DsRed1–IRES–eGFP–2A–rtTA, or TREAutoR3, using fluorescence microscope and Western blot. Similar to cells infected with TREAutoR3, the basal expression of both eGFP and DsRed1 in cells infected with our vectors is very low (Fig. 4). When these transduced 293T cells were exposed to media containing 1 μg/mL doxycycline for 48 H, the induction of the transgenes on all three vectors were similar too, suggesting our vectors did not alter the low basal expression and high efficiency of the template TREAutoR3 vector.
3.3. Induction of the tricistronic vectors in vivo Next we set out to investigate whether the tricistronic vectors we created can be induced in vivo, using a mouse xenograft model of A375 malignant melanoma cell line. A375 cells were transduced with either DsRed1–2A–eGFP–IRES–rtTA3 or DsRed1–IRES–eGFP–2A–rtTA3 vectors at a multiplicity of infection of 60 and injected subcutaneously into the left-flank regions of nude mice to generate tumors. These mice were then fed with water with or without doxycycline (200 μg/mL)
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for 3 weeks before the tumors were excised. The expression of both eGFP and DsRed1 were clearly induced by doxycycline in the A375 xenograft transduced with both vectors (Fig. 5), suggesting these two vectors can be efficiently induced in vivo.
4. Discussion The IRES and 2A sequences are both widely reported nucleotide elements that are capable of initiating translation in the middle of mRNA. However, different from the IRES sequence, the 2A sequence cannot recruit ribosome on its own, and its function relies on the translation machinery started by the transgene before it. Therefore, when the 2A sequence is used to separate genes, the stop codon of the gene before the 2A sequence has to be removed. In this case, the peptide encoded by the 2A sequence (except the last proline) will be added to the end of the upstream transgene, and an extra proline will be added to the beginning of the downstream gene [17, 18]. Moreover, it has also been published that the separation effect of the 2A sequence is not complete, and it would allow the production of around 20% fusion proteins [22]. In the current work, a weak fusion band of DsRed1 and eGFP can also be observed when the 2A sequence is placed between them (Fig. S1), supporting the existence of fusion proteins created by the 2A sequence. Although the fusion proteins created by the 2A sequence contain the full sequence of both exogenous proteins, they may not maintain the functions of these proteins. For example, compared with DsRed1–2A–eGFP–IRES–rtTA, the sensitivity to and the induction speed of DsRed1–IRES–eGFP–2A–rtTA by doxycycline is slightly weaker (Figs. 2 and 3). This is probably due to the 2A-sequence-mediated production of dysfunctional eGFP–rtTA3 fusion proteins that can no longer cooperate with doxycycline or activate RNA polymerase to initiate transcription. More functional rtTA3 proteins in cells transduced with DsRed1–2A–eGFP–IRES–rtTA led to higher doxycycline sensitivity and faster induction. Therefore, IRES may be more suitable to be placed before rtTA3 for efficient
All-in-One Multicistronic Tet-On Lentiviral Vectors
FIG. 3
FIG. 4
Induction of DsRed1 and eGFP in response to a titration of doxycycline treatment in 293T cells transduced with the tricistronic vectors. 293T cells transduced with (A) DsRed1–2A–eGFP–IRES–rtTA3 or (B) DsRed1–IRES–eGFP–2A–rtTA3 were treated with the indicated concentrations of doxycycline (0, 50, 100, 200, 500, and 1000 ng/mL in the media) for 48 H before the harvest. Expression of DsRed1 and eGFP proteins was analyzed using Western blot. Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panels of both (A) and (B).
Comparison of the TREAutoR3 template vector with the tricistronic vectors. 293T cells transduced with the indicated vectors were treated with or without doxycycline (1 μg/mL in the media) for 48 H and subjected to (A) fluorescence microscope or (B) Western blot examinations for the expression of DsRed1 and eGFP proteins. (A) Scale bars = 100 μm. (B) Relative protein expression of DsRed1 and eGFP was balanced by actin and is presented in the lower panel.
doxycycline induction. However, in the DsRed1–2A–eGFP– IRES–rtTA transduced cells, both DsRed1 and eGFP proteins contained extra amino acids and fusion proteins were produced (Fig. S1). Therefore, DsRed1–2A–eGFP–IRES–rtTA is probably not suitable for the expression of proteins such as mouse double minute 2 homolog in which both the C-terminus and
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the N-terminus have important roles in regulating protein modification and interaction, but may be better for proteins such as death-associated protein kinase 1 whose terminal regions are not reported for critical regulatory roles [23]. An important problem for inducible system is leakiness, which is due to the unexpected translation of rtTA3 in
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FIG. 5
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Induction of DsRed1 and eGFP in response to doxycycline treatment in mouse xenografts transduced with the tricistronic vectors. A375 cells were transduced with either DsRed1–2A–eGFP–IRES–rtTA3 or DsRed1–IRES–eGFP–2A–rtTA3. Cells were implanted subcutaneously into the left-flank regions of nude mice (n = 8). The mice were then fed with water with or without doxycycline (200 μg/mL) continuously for 3 weeks before tumor excision. DsRed1 and eGFP induction were detected on frozen tumor sections through fluorescence confocal microscope. Scale bar = 100 μm.
All-in-One Multicistronic Tet-On Lentiviral Vectors
transduced cells when doxycycline is not present. Although both of our vectors have similar low basal expression compared with the template TREAutoR3 (Fig. 4), weak leakage of eGFP and DsRed1 proteins can still be observed in Western blot and fluorescence staining (Figs. 2–4). However, the expression of the transgenes from leakage was insignificant in comparison with when the transduced cells were exposed to doxycycline (Figs. 2–4). Therefore, unless the transgenes can affect cellular processes at very low level, our vectors should be suitable for most target genes in biological research. Nevertheless, it will be useful to have a better promoter other than TRE that would allow further reduction or even elimination of leakiness in the inducible system in the future. In addition, the sequence of the target genes may significantly affect the efficiency of translation [24]. In the current work, we only tested our vectors by using GFP and DsRed1 genes, which are not present in mammalian cells. However, when other “genes of interest” are applied in the future application of these vectors, there may be unexpected activation or silencing of the exogenous genes, caused by their endogenous regulators or codon usage bias. Recently, Stergachis et al. have shown that the coding region may also regulate the binding of transcription factors [25]. Therefore, more work will be needed to validate the further application of these vectors. In conclusion, we have created two all-in-one Tet-On tricistronic vectors expressing two trangenes under the control of a single TRE promoter. Similar to the template TREAutoR3 vector, both new vectors displayed low basal expression level and efficient doxycycline induction in vitro and in vivo, suggesting that they can be used as useful tools for inducible epigenetic gene expression in cells and animals. However, on the basis of the positions of the 2A and IRES sequences that are used to separate the two transgenes and rtTA3, each new vector has its advantages. Further application of these constructs may shed light on discovering more characteristics of the vectors and the translation machinery of the IRES and 2A sequences.
5. Acknowledgments This work was supported by the National Natural Science Foundation of China for Young Scholar (grant no. 31301172)
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and the Foundation of Fujian Educational Committee (grant no. JA12061). The authors have no conflict of interest.
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