The authors thank S-Y Choi (Chonnam National University) for valuable comments and thorough reading of our manuscript. This work was supported by a research fund of Chungnam National University.

1. Transgene Subcloning
<ol>
	<li>Choose an appropriate restriction enzyme recognition site (or sites) in the MCS of pEUI(+), and subclone the gene of interest in the desired direction (Figure 1). 
	NOTE: The EGFP or FLAG epitope-tagged ankyrin repeat domain 13A (ANKRD13A) transgene was subcloned in EcoRI site of the pEUI(+) vector using T4 DNA polymerase in a sequence- and ligation-independent cloning method19.</li>
	<li>Linearize the pEUI(+) with EcoRI for 1 h at 37 &#176;C. Mix 50 ng/&#181;L of linearized pEUI(+) with 40 ng/&#181;L of PCR-amplified EGFP or ANKRD13A, and react with T4 DNA polymerase (1 U) for 1 min at room temperature (RT) before incubation on ice for 10 min. Add 10 &#181;L of the reaction mixture directly to 100 &#181;L of DH10B competent cells for the following transformation experiment. 
	NOTE: Although several restriction enzyme recognition sites were added to the MCS of pEUI(+) (Figure 1B), we recommend subcloning the target gene into the EcoRI site, using ligation-independent cloning technology19,20.</li>
	<li>Verify the insert by sequencing and then purify the DNA using a DNA purification kit that is suitable for transfection.</li>
</ol>
2. Transient Transfection and Teb Treatment
<ol>
	<li>Prepare four cell culture dishes (60 mm diameter) containing freshly passaged HEK293 cells in 5 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5 - 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin, 100 &#181;g/mL streptomycin). Hereafter, &#34;cell culture medium&#34; or simply &#34;culture medium&#34; represents the DMEM supplemented with FBS and antibiotics. Maintain the HEK293 cells at 37 &#176;C.</li>
	<li>Transfect 1 &#181;g of purified DNA into the HEK293 cells at 50 - 60% density using 3 &#181;L of transfection reagent. Add the transfection reagent to 200 &#181;L of diluent (DMEM without FBS and antibiotics) containing the DNA. After the incubation of the reaction mixture for 30 min at RT, add the mixture to the HEK-293 cells on the culture dish. Use two dishes for transfection, and leave the other two untreated as controls.</li>
	<li>After 12 - 24 h of transfection, add Teb (final concentrations, 0.2 -&#160;10 &#181;mol) to one of the transfected samples and to one of the non-transfected controls. Leave the other two samples untreated as experimental controls. 
	NOTE: The Teb stock solution (50 mM dissolved in dimethyl sulfoxide) can be diluted in cell culture medium, DMEM, or phosphate-buffered saline (PBS) at an appropriate concentration before adding to the cultured cells depending on the experimental parameters. However, due to the low solubility of Teb in aqueous solution, avoid preparing a master mix with a high concentration of Teb in medium; instead, replace the complete cell culture medium with fresh medium containing Teb at the desired concentration.</li>
	<li>Cultivate the Teb-treated and untreated cells for 12 - 48 h at 37 &#176;C.</li>
	<li>If using the EGFP insert, analyze EGFP expression under a fluorescent microscope ( Figure 2). Otherwise, harvest the cells for an immunoblotting assay.
	<ol>
		<li>For the immunoblotting assay, rinse the cells twice with ice-cold PBS and then scrape them out using a cell scraper.</li>
		<li>For piling the cells, spin down the harvests in a 15 mL conical tube at 21,000 x g for 2 min at 18-25 &#176;C (RT).</li>
		<li>After discarding the PBS completely, add 0.5 mL of ice-cold mammalian protein extraction reagent (M-PER), together with protease inhibitor (5 &#181;L of 100x&#160;protease inhibitor cocktail), to the pellet if the cells are cultured in a 60 mm dish.</li>
		<li>Re-suspend the cells with several rounds of pipetting, and then incubate the suspended cells for 15 min at 18 - 25 &#176;C (RT).</li>
		<li>Spin the cell lysate at 21,000 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the aqueous supernatant into a fresh 1.5 mL microcentrifuge tube.</li>
		<li>Analyze the supernatant by immunoblotting, following standard protocols with appropriate antibodies.</li>
	</ol>
	</li>
</ol>
3. Generation of Stable HEK293 Cell Lines with Genomic Integration of the pEUI(+) Gene Switch
<ol>
	<li>Transfect 5 - 10 &#181;g of the purified pEUI(+) with the transgene into HEK293 cells that are 50 - 60% confluent in a 90 mm diameter cell culture dish, using 15 - 30 &#181;L of the transfection reagent. Add the transfection reagent to 500 &#181;L of diluent (DMEM without FBS and antibiotics) containing the DNA. After the incubation of the reaction mixture for 30 min at RT, add the mixture to HEK-293 cells on the culture dish.</li>
	<li>After the transfection, allow the transfectants to grow and express the puromycin-resistance gene products under non-selective culture medium (without puromycin) for 24 - 48 h at 37 &#176;C.</li>
	<li>Dissociate the cells from the culture dishes by trypsinization. After discarding the culture medium, rinse the transfectants with pre-warmed PBS (37 &#176;C), add 1 mL of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA), and incubate for 1 min at 37 &#176;C. After quenching the trypsin/EDTA by adding pre-warmed culture medium (10 mL, 37 &#176;C), harvest the cells using a cell scraper, followed by centrifugation in a 15 mL conical tube at 21,000 x g for 2 min at 18-25&#176;C.</li>
	<li>Re-suspend the transfectants in 50 mL of pre-warmed culture medium (37 &#176;C), and transfer 10 mL of the cells into 90 mm fresh cell culture dishes. Culture the cells at 37 &#176;C until 50 - 60% confluency before the puromycin treatment (in general, this takes approximately 24 - 48 h).</li>
	<li>Cultivate the transfectants at 37 &#176;C in cell culture medium supplemented with 3 &#181;g/mL puromycin for 3 - 4 weeks. During the selection period, change the cell culture medium containing puromycin (3 &#181;g/mL) every 2 -&#160;3 days to maintain selection pressure. When the cells become fully confluent, split them following the procedures in steps 3.3 - 3.4, with puromycin-containing culture medium. Discard the remaining cells in the culture medium, if not needed. 
	NOTE: Puromycin selection conditions must be established experimentally for specific cell types. We used 3 &#181;g/mL of puromycin in cell culture medium; this was enough to induce cell death in non-transfected HEK293.</li>
	<li>Harvest the cells from individual colonies, using a cloning cylinder, and follow the manufacturer&#39;s instructions.
	<ol>
		<li>Discard the cell culture medium. Rinse the plate twice with pre-warmed (37 &#176;C) PBS to remove floating cells.</li>
		<li>Using sterile forceps, set the cloning cylinder over the individual colonies.</li>
		<li>Add 0.2 mL of 0.35% trypsin/EDTA to each cylinder.</li>
		<li>Incubate the dish at 37 &#176;C for 1 min until the cells are detached, and then add a few drops of pre-warmed (37 &#176;C) culture medium to the cylinders.</li>
		<li>Transfer the cells from each cylinder to individual wells of a 6-well plate, with 2 mL of culture medium and 1 &#181;g/mL of puromycin in each well. 
		NOTE: When the cells become confluent, scale up the culture. Now clone(s) may be established.</li>
	</ol>
	</li>
	<li>Maintain individual clones in cell culture medium supplemented with 1 &#181;g/mL of puromycin.</li>
	<li>Validate individual colonies by testing their sensitivity to Teb treatment (final concentration, 10 &#181;M) for 24 h. Test the responsiveness of the clones to the Teb by analyzing the expression level of the transgene with immunoblotting. Select a desired clone (or clones). Cultivate a Teb-treated and an untreated sample in culture medium for 12 - 48 h at 37 &#176;C before analyzing the induction level of the transgene. The expression level of transgene can be measured through immunoblotting, following the procedures in steps 2.5.1 - 2.5.7.</li>
</ol>
4. Depletion of Teb
<ol>
	<li>Quench the Teb stimuli by replacing Teb containing cell culture medium with Teb-free medium. 
	NOTE: It is recommended to replace the growth media without first reseeding the cells onto a new culture dish. Residual Teb appears to cling to the plastic dish and will elicit a strong background expression of the transgene. Before reseeding the cells on a new culture plate, rinse the harvested cells several times with either culture medium or PBS.
	<ol>
		<li>Dissociate the cells from the culture dishes by trypsinization. After discarding the Teb containing culture medium, rinse the Teb-stimulated cells twice with pre-warmed PBS (37 &#176;C), add 1 mL of 0.25% trypsin/EDTA, and incubate for 1 min at 37 &#176;C. After quenching the trypsin/EDTA by adding pre-warmed Teb-free culture medium (10 mL, 37 &#176;C), harvest the cells using a cell scraper, followed by centrifugation in a 15 mL conical tube at 21,000 x g for 2 min at 18-25 &#176;C.</li>
		<li>Re-suspend the cells in 50 mL of pre-warmed Teb-free culture medium (37 &#176;C), and transfer 5 mL of the cells into three 60 mm fresh cell culture dishes. Culture the cells at 37 &#176;C for 24, 48, and 72 h respectively.</li>
		<li>Harvest the cells at different time points and measure the expression level of the transgene by using immunoblotting. 
		NOTE: Under these experimental conditions, the transgene expression became undetectable approximately 3 days post-culture of the cells in the Teb-free cultural medium (Figure 3). The expression level of transgene can be measured by immunoblotting, as per steps 2.5.1 - 2.5.7.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Rossi, F. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+inducible+gene+expression+systems.">Recent advances in inducible gene expression systems.</a> Curr. Opin. Biotechnol. 9 (5), 451-456 (1998).</li><li>Clackson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+gene+expression+systems.">Regulated gene expression systems.</a> Gene Ther. 7 (2), 120-125 (2000).</li><li>Suzuki, Y., Suzuki, Y., Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatable+lentiviral+system.">Gene regulatable lentiviral system.</a> Viral Gene Therapy. , 285-309 (2011).</li><li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters.">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proc. Natl. Acad. Sci. USA. 89 (12), 5547-5551 (1992).</li><li>Baron, U., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+repressor-based+system+for+regulated+gene+expression+in+eukaryotic+cells:+principles+and+advances.">Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances.</a> Methods Enzymol. 327, 401-421 (2000).</li><li>Hentges, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP/mTOR+is+required+for+proliferation+and+patterning+during+embryonic+development+in+the+mouse.">FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse.</a> Proc Natl Acad Sci U S A. 98 (24), 13796-13801 (2001).</li><li>Banaszynski, L. A., Liu, C. W., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FKBP.rapamycin.FRB+ternary+complex.">Characterization of the FKBP.rapamycin.FRB ternary complex.</a> J Am Chem Soc. 127 (13), 4715-4721 (2005).</li><li>Seto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+and+mTOR:+a+serendipitous+discovery+and+implications+for+breast+cancer.">Rapamycin and mTOR: a serendipitous discovery and implications for breast cancer.</a> Clin Transl Med. 1 (1), 29 (2012).</li><li>Ulmann, A., Peyron, R., Silvestre, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+uses+of+mifepristone+(MFP).">Clinical uses of mifepristone (MFP).</a> Ann N Y Acad Sci. 761, 248-260 (1995).</li><li>Sitruk-Ware, R., Spitz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+properties+of+mifepristone:+toxicology+and+safety+in+animal+and+human+studies.">Pharmacological properties of mifepristone: toxicology and safety in animal and human studies.</a> Contraception. 68 (6), 409-420 (2003).</li><li>Yao, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+ecdysone+receptor+is+the+product+of+EcR+and+ultraspiracle+genes.">Functional ecdysone receptor is the product of EcR and ultraspiracle genes.</a> Nature. 366 (6454), 476-479 (1993).</li><li>Riddiford, L. M., Cherbas, P., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+receptors+and+their+biological+actions.">Ecdysone receptors and their biological actions.</a> Vitam Horm. 60 (2000), 1-73 (2000).</li><li>Saez, E., Nelson, M. C., Eshelman, B., Banayo, E., Koder, A., Cho, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ligands+and+coligands+for+the+ecdysone-regulated+gene+switch.">Identification of ligands and coligands for the ecdysone-regulated gene switch.</a> Proc Natl Acad Sci USA. 97 (26), 14512-14517 (2000).</li><li>Karzenowski, D., Potter, D. W., Padidam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+control+of+transgene+expression+with+ecdysone+receptor:+gene+switches+with+high+sensitivity+robust+expression,+and+reduced+size.">Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity robust expression, and reduced size.</a> Biotechniques. 39 (2), 191-200 (2005).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+receptor-based+singular+gene+switches+for+regulated+transgene+expression+in+cells+and+adult+rodent+tissues.">Ecdysone receptor-based singular gene switches for regulated transgene expression in cells and adult rodent tissues.</a> Mol. Ther. Nucleic Acids. 5 (9), e367 (2016).</li><li>Esengil, H., Chang, V., Mich, J. K., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+regulation+of+zebrafish+gene+expression.">Small-molecule regulation of zebrafish gene expression.</a> Nat Chem Biol. 3 (3), 154-155 (2007).</li><li>Padidam, M., Gore, M., Lu, D. L., Smirnova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-inducible,+ecdysone+receptor-based+gene+expression+system+for+plants.">Chemical-inducible, ecdysone receptor-based gene expression system for plants.</a> Transgenic Res. 12 (1), 101-109 (2003).</li><li>Knopf, F., Schnabel, K., Haase, C., Pfeifer, K., Anastassiadis, K., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dually+inducible+TetOn+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetOn systems for tissue-specific conditional gene expression in zebrafish.</a> Proc Natl Acad Sci USA. 107 (46), 19933-19938 (2010).</li><li>Jeong, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+sequence-+and+ligation-independent+cloning+as+a+rapid+and+versatile+cloning+method+for+functional+genomics+studies.">One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies.</a> Appl Environ Microbiol. 78 (15), 5440-5443 (2012).</li><li>Zhu, B., Cai, G., Hall, E. O., Freeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-fusion+assembly:+seamless+engineering+of+multidomain+fusion+proteins,+modular+vectors,+and+mutations.">In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations.</a> Biotechniques. 43 (3), 354-359 (2007).</li><li>No, D., Yao, T. P., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone-inducible+gene+expression+in+mammalian+cells+and+transgenic+mice.">Ecdysone-inducible gene expression in mammalian cells and transgenic mice.</a> Proc Natl Acad Sci U S A. 93 (8), 3346-3351 (1996).</li><li>Sawada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+insecticidal+activity+of+benzoheterocyclic+analogues+of+N'-benzoyl-N-(tert-butyl)benzohydrazide:+Part+1.+Design+of+benzoheterocyclic+analogues.">Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 1. Design of benzoheterocyclic analogues.</a> Pest Manag Sci. 59 (1), 25-35 (2003).</li><li>Lafont, R., Girault, J. P., Kerb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excretion+and+metabolism+of+injected+ecdysone+in+the+white+mouse.">Excretion and metabolism of injected ecdysone in the white mouse.</a> Biochem Pharmacol. 37 (6), 1174-1177 (1988).</li><li>Carlson, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+and+biological+properties+of+methoxyfenozide,+a+new+insecticidal+ecdysteroid+agonist.">The chemical and biological properties of methoxyfenozide, a new insecticidal ecdysteroid agonist.</a> Pest Manag Sci. 57 (2), 115-119 (2001).</li><li>McConnell, M. J., Imperiale, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+adenovirus+and+its+use+as+a+vector+for+gene+therapy.">Biology of adenovirus and its use as a vector for gene therapy.</a> Hum Gene Ther. 15 (11), 1022-1033 (2004).</li><li>Gorell, E., Nguyen, N., Lane, A., Siprashvili, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+skin+diseases.">Gene therapy for skin diseases.</a> Cold Spring Harb Perspect Med. 4 (4), a015149 (2014).</li><li>Kotterman, M. A., Chalberg, T. W., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Vectors+for+Gene+Therapy:+Translational+and+Clinical+Outlook.">Viral Vectors for Gene Therapy: Translational and Clinical Outlook.</a> Annu Rev Biomed Eng. 17, 63-89 (2015).</li><li>Matsumoto, T., Yamaguchi, M., Kuzume, M., Matsumiya, A., Kumada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+gene+transfer+with+adenovirus+vector+via+the+spleen+safely+and+effectively+improves+posthepatectomized+conditions+in+diabetic+rats.">Insulin gene transfer with adenovirus vector via the spleen safely and effectively improves posthepatectomized conditions in diabetic rats.</a> J Surg Res. 110 (1), 228-234 (2003).</li><li>Han, J., McLane, B., Kim, E. H., Yoon, J. W., Jun, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remission+of+diabetes+by+insulin+gene+therapy+using+a+hepatocyte-specific+and+glucose-responsive+synthetic+promoter.">Remission of diabetes by insulin gene therapy using a hepatocyte-specific and glucose-responsive synthetic promoter.</a> Mol Ther. 19 (3), 470-478 (2011).</li></ol>

We have no conflicts of interest related to this report.

The main drawback of using a binary gene expression switch is the need to simultaneously deliver two separate plasmids (driver and effector) into the target cell. This can lead to an unequal distribution of plasmids and result in an inconsistent response of the switch to the inducers1,2,3. Another shortcoming of the two-vector system is that a minimum of two rounds of antibiotic selection are required to identify promising cell ...

Several different gene switches have been explored for their ability to precisely regulate transgene expression in cell culture and animal models, with varying degrees of success1,2,3. Potential gene switch systems should meet several stringent criteria, including: precise directional expression of the transgene, dosage- and time-dependent responsiveness to inducers, negligible leakiness of the promoter, reversibility of transgene activation through inducer removal, and inducer toxicity levels tolerable to cell lines and laboratory animals1,2,3. Several small molecule inducers have been exploited, including tetracycline4,5, rapamycin6,7,8, mifepristone9,10, and ecdysone11,12,13,14. In general, these systems require at least two plasmids containing two or three discrete expression units, one or two of which compose a regulatory element (inducer plasmid) that binds a small molecule inducer to gain transcriptional activity; and another plasmid (effector plasmid) containing the transgene under the control of a regulatory DNA region that allows the access of the inducer-bound regulatory element. The main drawback of the binary system is that it requires the concomitant introduction of two vectors (inducer and effector) into the target cell. In transient transgene expression experiments, the simultaneous transfection of two plasmids inevitably produces a population of cells that is singly transfected with either the inducer or the effector, or co-transfected unequally. In addition, the binary system requires at least two rounds of antibiotic selection to establish stable cell lines, which is time-consuming and laborious. Thus, combining all of the components required for transgene expression and regulation into a single vector would be ideal to guarantee the concomitant expression of all the required elements in a single cell and allow for the regulation of transgene expression through treatment with small molecule inducers.
To overcome the shortcomings of binary gene switches, we recently developed a singular gene switch, using a Drosophila melanogaster EcR-based gene inducible system15. Ecdysone mediates gene expression when it binds to the EcR/ultraspiracle (USP) heterodimer, which in turn induces binding of the EcR to DNA regulatory elements3,11. The vertebrate retinoid X receptor (RXR), an ortholog of insect USP, recruits EcR to form an active transcriptional activator16. Thus, for the targeted expression of a transgene in vertebrate cells or tissue, the EcR and RXR must be co-expressed simultaneously before being stimulated by ecdysone or its agonists. Since the heterodimeric feature of the EcR-based gene switch can be influenced by the endogenous RXR level, Padidam et al. replaced the EcR DNA-binding and activation domains with heterologous GAL4 DNA-binding, herpes simplex virus protein vmw65 (VP16) activation domains fused to the EcR ligand-binding domain, which then became unresponsive to endogenous RXR and formed a homodimer to gain transcriptional activity17. The EcR-based gene switch was further improved by constructing a chimeric protein comprised of minimal VP16 activation domain combined with GvEcR, which formed a homodimer and localized in the cytosol in the absence of the ecdysone agonist, Teb16,18. By binding to Teb, the GvEcR changed its subcellular localization from the cytosol to the nucleus to recognize a hybrid promoter comprised of zebrafish E1b minimal promoter combined with a ten tandem repeated upstream activation sequences (UASs) to initiate the transcription of the target gene16.
To simplify the previously reported EcR-based gene switches16,18, we combined the binary features of the system into a single vector equipped with all the required elements for stimulating transgene expression, and then designated the newly generated vector, pEUI(+) (GenBank accession number: KP123436, Figure 1A)15. The effector responding to the GvEcR driver (hereafter driver) consists of ten tandem UAS repeats next to an E1b minimal promoter followed by a multiple cloning site (MCS) with EcoRI, PmlI, NheI, BmtI, AfeI, and AflII recognition sequences (Figure 1B). Additionally, to facilitate the selection of transfected cells, an SV40 promoter-driven puromycin N-acetyl-transferase (PAC) gene was placed between the driver and effector regions to maintain stable cell lines (Figure 1).
We describe a detailed protocol for the transient and stable modulation of transgene expression using pEUI(+). In addition, we provide detailed instructions for the successful use of Teb as an ecdysone agonist.

<table><tbody><tr><td>pEUI(+)</td><td>TransLab</td><td></td><td>The patent license was transferred to TransLab Inc.&amp;nbsp;</td></tr><tr><td>Gene-Fect Transfection Reagent</td><td>TransLab</td><td>TLC-001</td><td></td></tr><tr><td>HEK293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>Tebufenozide</td><td>Fluka</td><td>31652</td><td></td></tr><tr><td>Cloning cylinder</td><td>Sigma</td><td>CLS31668</td><td></td></tr><tr><td>Puromycin</td><td>Corning</td><td>58-58-2</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Welgene</td><td>LS015-01</td><td></td></tr><tr><td>DMEM</td><td>Welgene</td><td>LM001-05</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Welgene</td><td>S001-01</td><td></td></tr><tr><td>Cell culture dish</td><td>SPL life science</td><td>11090</td><td></td></tr><tr><td>mouse FLAG M2</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>anti-alpha tubulin antibody</td><td>Calbiochem</td><td>CP06</td><td></td></tr><tr><td>Cloning cylinder</td><td>Sigma</td><td>CLS31668</td><td></td></tr><tr><td>NucleoBond Xtra Midi</td><td>Macherey-Nagel</td><td>740410.1</td><td></td></tr><tr><td>M-PER Mammalian Protein&lt;br/&gt; Extraction Reagent</td><td>Thermo Fisher</td><td>78505</td><td></td></tr><tr><td>100X Protease inhibitor Cock. III</td><td>T&amp;amp;I</td><td>BPI-9200</td><td></td></tr></tbody></table>

an ecdysone receptor-based singular gene switch for deliberate expression of transgene with robustness, reversibility, and negligible leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

The GvEcR-based singular gene switch is depicted in Figure 1A. The pEUI(+) vector was optimized for regulated transgene expression by the treatment with Teb. The effector region of pEUI(+) comprises a 10xUAS and an E1b minimal promoter followed by an MCS containing EcoRI, PmlI, NheI, BmtI, AfeI, and AflII restriction enzyme recognition sites. An SV40 poly(A) signal was added behind the MCS (Figure 1B). A PAC gene was ins...

Watch this Scientific Journal Video about An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness at JoVE.com

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

an-ecdysone-receptor-based-singular-gene-switch-for-deliberate

Research

JoVE Journal

Genetics

6.4K Views.  Chungnam National University. Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment. 

Video: An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Inducible and Reversible Dominant-negative DN Protein Inhibition

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Developmental Biology

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Immunology and Infection

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Bioengineering

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Biology

Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System

Source: Migneault, F. et al, <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/60654">Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells.</a> J. Vis. Exp. (2020).
This video describes the method of regulating gene expression in cultured cells by transfection with a plasmid containing a gene of interest and a regulatory plasmid. The expression of this plasmid can be controlled by tetracycline or its synthetic analogs, making this system a useful model to study regulatable gene expression.

Source: Migneault, F. et al, Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Summary

Explore More Videos

Chapters in this video

Controllable Ion Channel Expression through Inducible Transient Transfection

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System