Tetracycline (Tet) Inducible Expression

Inducible and repressible promoters are powerful tools to control gene expression and study biological targets. The tetracycline (tet) system is one of the most popular choices for rapid, efficient, and reversible control of gene expression in eukaryotes.

To avoid secondary effects from repurposing endogenous systems that respond to natural stimuli like hormones or metal ions, scientists have adapted an antibiotic resistance operon from E. coli: the tetracycline repressor protein (TetR) and operator (tetO, a 19 nucleotide sequence, TCCCTATCAGTGATAGAGA). In normal conditions, the TetR protein binds to tetO, blocking transcription of the downstream gene. If tet or one of its analogs like doxycycline (dox) is present, TetR binds to tetracycline and releases tetO, enabling transcription (Figure 1).

While TetR and tetO could be a basic tool to control gene expression, the system has been engineered with a variety of improvements. A synthetic Tet Response Element (TRE) typically features seven copies of tetO upstream of a minimal CMV promoter, and other tet- or dox-dependent promoters (sometimes generally called Ptet) have been designed by combining tetO sequences with elements like the H1 RNA promoter. And, most importantly, the TetR protein has been modified to provide two classes of tet-responsive systems: Tet-Off and Tet-On.

Read on to learn more about the components of tet systems and how to use them, or skip ahead to view highlighted Tet plasmids.

Figure 1: Tet-regulated expression systems. Left: natural TetR mechanism; center: Tet-Off; right: Tet-On. TRE: Tet response element; tTA: tetracycline-controlled transactivator (fusion of TetR with VP16 transcriptional activation domain); rtTA: reverse tetracycline-controlled transactivator (fusion of mutated TetR with VP16 transcriptional activation domain). For simplicity, other promoter elements are not shown.

Tetracycline Off (Tet-Off)

The first major advance was the Tet-Off system. A tetracycline-controlled transactivator (tTA) was created by fusing TetR with the activation domain of the herpes simplex virus VP16 protein (Gossen and Bujard, 1992). This work also introduced the TRE, placing seven copies of tetO upstream of the minimal CMV promoter.

In the absence of tetracycline, tTA binds to the TRE and its VP16 domain promotes gene expression. In the presence of tetracycline, tTA unbinds from the TRE, resulting in reduced gene expression. Tetracycline Off is also known as the tTA-dependent or tet-repressible system.

Tetracycline On (Tet-On)

In 1995, Gossen et al. used random mutagenesis to identify the amino acid residues of TetR that were important for tetracycline-dependent repression. Mutating these residues led to a reverse Tet repressor, or rTetR, which specifically binds the TRE in the presence of tetracycline. A new reverse tetracycline-controlled transactivator (rtTA) was created by fusing rTetR with VP16.

In the absence of tetracycline, rtTA is unable to bind the TRE. In the presence of tetracycline, rtTA binds the TRE, promoting gene expression. Tetracycline On is also known as the rtTA-dependent or tet-inducible system.

In effect, Tet-On is similar to the natural TetR-tetO system, although the DNA-binding profile is reversed (Figure 1). However, rtTA can induce stronger expression of the downstream gene via its activation domain than simply removing the repressor.

2nd and 3rd Generation Systems

Subsequent efforts have improved the components of these systems for better efficiency, sensitivity, and control of expression. These include:

• Tet-On Advanced: improved transactivator (rtTA-Advanced, also known as rtTA2^S-M2) with greater sensitivity to doxycycline and three copies of a minimal activation domain from VP16 for increased induction of gene expression and reduced side effects (Urlinger et al., 2000).
• rtTA3 and Tet-On 3G: further improved rtTA variants with lower background activity and greater sensitivity to doxycycline (Zhou et al., 2006). Tet-On 3G systems can achieve >10,000-fold induction of the gene of interest.
• Tight TRE promoter or P_Tight: reduced background expression and tighter binding of Tet-Advanced transactivator (developed by Clontech, based on Resnitzky et al., 1994).
• TRE3G promoter: also optimized into P_TRE3GV for viral expression and P_TRE3GS for all-in-one systems (Loew et al., 2010; Heinz et al., 2011), engineered to eliminate binding of endogenous mammalian transcription factors to further minimize background expression.
• Improved expression of the transactivator proteins in mammalian cells or other species by codon optimization of their sequence and eliminating interactions with endogenous factors.

Experimental Considerations

Expression System

• If your gene of interest should only be expressed occasionally, use Tet-On: express an rtTA or TetR and include a tet-responsive promoter upstream of your gene of interest.
• If your gene of interest should be generally active and only turned off occasionally, use Tet-Off: express a tTA and include a tet-responsive promoter upstream of your gene of interest.
• Many plasmids use "TetO" or "Tet-O" in their name or description, meaning they contain a TRE or tetO and can serve as either Tet-Off or Tet-On systems, depending on whether they are used alongside a tTA or rtTA, respectively.

You should also choose between:

• Dual vector system: express the transactivator and the tet-controlled gene of interest from two separate vectors. Stable cell lines or transgenic animals can be made to achieve greater control over expression with flexibility for your experiments. For example, you could integrate a transactivator under a tissue-specific promoter and administer the transgene separately.
• All-in-one system: express both the transactivator and the tet-controlled gene of interest from one plasmid. A single vector approach may not have as much flexibility and control as a dual vector design, but it is easier to implement and ensures that all transfected or transduced cells express both components in the same relative ratio.

Different generations of transactivators and promoters are generally cross-compatible. Your choice of transactivator, promoter, and type of transduction (transient or stable) may depend on the desired sensitivity, level of induction, or amount of background expression. For example, only a relatively low level of doxycycline can access the brain so a highly sensitive transactivator may be needed. Or, when inducing a potentially toxic protein you may need extremely minimal background expression and only moderate induction. See Das et al., 2016 for comparison of Tet-On systems in different applications.

Use of Tetracycline or a Derivative

Doxycycline, a derivative of tetracycline, is a preferred effector for tet-responsive systems. Dox binds with high affinity to TetR and derivatives like rTetR, rtTA, and tTA. Dox has good tissue distribution, low toxicity, a known half-life (24 hours), and is relatively inexpensive. The induction profiles for dox and other tetracycline derivatives can be found in Krueger et al., 2004. Some cell culture sera may contain tetracycline or its derivatives which can result in unintended induction. Check the manufacturer’s details to know whether your serum is tetracycline-free.

Highlighted Plasmids

Browse the tables below to find popular empty backbones, expression vectors for transactivators, and vectors for other applications, or search our collection for all dox-regulated plasmids.

Looking for viral preps? Find in-stock viral preps of tetracycline transactivators and inducible tools or consider our AAV Packaged on Request service available for many transactivator or tet-inducible vectors.

Tetracycline-inducible Backbones

Find backbones or constructs to use for cloning to control expression of your gene of interest.

	ID	Plasmid	Description	Transactivator	Promoter	PI
	21915	Tet-pLKO-puro	Lentiviral Tet-On plasmid for inducible expression of shRNA with puromycin selection. See Plasmid #21916 for neomycin selection.	TetR	H1-2O2	Dmitri Wiederschain
	85966	EZ-Tet-pLKO-Puro	Lentiviral Tet-On plasmid for easier cloning and design of inducible shRNA expression, with puromycin selection. See Plasmid #85973 for blasticidin and Plasmid #85972 for hygromycin selection.	TetR	H1-2O2	Cindy Miranti
	104321	tet-pLKO-sgRNA-puro	Lentiviral Tet-On plasmid for inducible expression of sgRNA	TetR	H1-2O2	Nathanael Gray
	35625	pAAV-Ptet-RFP-shR-rtTA	AAV Tet-On shRNA vector. To evaluate shRNA efficacy by fluorescence, use Plasmid #35626 as a reporter for cDNA target.	rtTA3	Ptet, miniCMV	Howard Gu
	44012	pInducer20	Lentiviral Tet-On vector for inducible expression. See article (Meerbrey et al., 2011) for additional tookit plasmids for shRNA or gene expression.	rtTA-Advanced	H1-2O2	Stephen Elledge
	111177	LT3GEPIR	Lentiviral Tet-On inducible RNAi with miR-E (miR-30 variant)-based backbone	rtTA-Advanced	TRE3G	Johannes Zuber
	60495	pSBtet-GP	Sleeping Beauty transposon system with luciferase in cloning site. See article (Kowarz et al., 2015) for additional selection markers.	rtTA-Advanced	Tight TRE	Eric Kowarz
	96930	XLone-GFP	Tet-On PiggyBac vector for inducble expression of EGFP	Tet-On 3G rtTA	TRE3GS	Xiaojun Lian
	171123	pLVX-TetOne-Puro-GFP	Lentiviral Tet-On vector for inducible expression of EGFP	Tet-On 3G rtTA	TRE3GS	Jason Sheltzer
	11651	pLVUT-tTR-KRAB	Lentiviral Tet-On vector for inducible expression of transgene and/or shRNA. See article (Szulc et al., 2006) for Tet-Off and additional plasmids.	tetR-KRAB	TRE	Patrick Aebischer, Didier Trono
	41393	pCW57.1	Lentiviral Tet-On vector for inducible expression; insert with Gateway cloning and puromycin selection	rtTA-Advanced	Tight TRE	David Root
	100521	pCW57.1-MAT2A	Lentiviral Tet-Off vector for repressible expression with blasticidin selection. *Note: this vector contains an insert (MAT2A) which can be replaced by another gene of interest.	tTA-Advanced	Tight TRE	David Sabatini
	19407	pTREtight2	Empty backbone for Tet-controlled expression (On or Off)	None	Tight TRE	Markus Ralser
	64238	pTet-IRES-EGFP	Lentiviral plasmid for Tet-controlled expression of transgene of interest with EGFP (On or Off)	None	TRE, miniCMV	Maria Lung
	16542	pBI-MCS-EGFP	Bidirectional promoter (Pbi) for Tet-responsive expression (On or Off) of both your gene of interest and EGFP. Pbi contains a TRE between two minimal CMV promoters.	None	Pbi (TRE, miniCMV)	Bert Vogelstein

Transactivators (tTA or rtTA)

Find a construct that expresses the transactivator for your tetracycline system.

	ID	Plasmid	Description	Transactivator	PI
	26429	pLenti CMV rtTA3 Blast (w756-1)	Lentiviral Tet-On vector with blasticidin selection. See Plasmid #26730 for hygromycin selection.	rtTA3	Eric Campeau
	128061	pLVX-Tet3G blasticidin	Lentiviral Tet-On vector with CMV promoter	Tet-On 3G rtTA	Oskar Laur
	96963	pCAG-TetON-3G	Mammalian expression of the Tet-On 3G transactivator	Tet-On 3G rtTA	Elena Cattaneo
	104543	PB-EF1a-TetOn3G	PiggyBac vector expressing Tet-On 3G transactivator constitutively expressed by EF1a promoter	Tet-On 3G rtTA	David Vereide
	120309	pAAV-FAH-rtTA3G	AAV vector to express Tet-On 3G transactivator under the c-Fos promoter	Tet-On 3G rtTA	Bong-Kiun Kaang
	20342	FUW-M2rtTA	Lentiviral Tet-On vector, expressing rtTA from UbC promoter	rtTA-Advanced	Rudolf Jaenisch
	25434	pMA2640	Retroviral Tet-On vector for CMV-driven rtTA with EGFP and Blasticidin selection	rtTA-Advanced	Mikhail Alexeyev
	17492	pLenti CMV TetR Blast (716-1)	Lentiviral Tet-On vector expressing TetR from CMV promoter	TetR	Eric Campeau, Paul Kaufman
	175274	pAAV-rtTA	AAV Tet-On vector with neuron-specific expression of rtTA3 from hSyn promoter	rtTA3	Wei Xu
	26803	pEnt L1L3 EF1a-tTA-2	Gateway entry vector to express tTA from EF1α promoter, for Tet-Off. See Plasmid #27106 for rtTA.	tTA	Edward Hsiao
	99118	pAAV-CAG-tTA	AAV Tet-Off vector, expresses tTA from the CAG promoter. See article (Chan et al., 2017) for other viral tTA plasmids.	tTA	Viviana Gradinaru
	104102	pCAG-tTA	Mammalian expression of tTA from the CAG promoter for Tet-Off	tTA-Advanced	Takeshi Imai
	104109	pAAV-Syn1-tTA	AAV Tet-Off vector, expressed tTA from the hSyn promoter	tTA-Advanced	Takeshi Imai

Other Tet Applications

Explore some other popular applications of tetracycline systems or search our collection for all dox-regulated plasmids.

	ID	Plasmid	Description	PI
	63800	PB-TRE-dCas9-VPR	Tet-inducible expression of SP-dCas9-VPR transcriptional activator with Tet-On 3G	George Church
	87360	TLCV2	Lentiviral vector for tet-inducible Cas9-2A-EGFP expression. Based on LentiCRISPR v2.	Adam Karpf
	167935	pLenti-tetON-KRAB-dCas9-DHFR-EF1a-TagRFP-2A-tet3G	Tet-inducible expression of KRAB-dCas9-DHFR for CRISPRi. See Plasmid #167937 for CRISPRa	Emma Rawlins
	72835	pMK243 (Tet-OsTIR1-PURO)	Expresses OsTIR1 under the control of a TRE3GS promoter for conditional auxin-inducible degron system	Masato Kanemaki
	92099	AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep	Tet-inducible expression gene of interest fused with 3xFLAG and 2xStrep-tag from TRE3G promoter for Tandem-Affinity Purification.	Yannick Doyon
	20321	TetO-FUW-OSKM	Tet-On inducible expression of mouse Oct4, Sox2, Klf4, and Myc for iPS cell generation	Rudolf Jaenisch
	51543	FUW-tetO-hOKMS	Tet-On inducible expression of human Oct4, Sox2, Klf4, and Myc for iPS cell generation	Tarjei Mikkelsen
	172115	PB-TO-hNGN2	Piggybac Tet-On plasmid for differentiating iPSCs into glutamatergic neurons via human NGN2 expression. See more from the iPSC Neurodegenerative Disease Initiative (iNDI) Collection.	Michael Ward
	105840	pUCM-AAVS1-TO-hNGN2	Introduction of dox-inducible human NGN2 into AAVS1 safe harbor site, for iPSC differentiation into cortical neurons	Michael Ward
	100798	pAAV-syn-FLEX-splitTVA-EGFP-tTA	Helper virus for monosynaptic tracing. To be coinjected with pAAV-TREtight-mTagBFP2-B19G (Plasmid #100799).	Ian Wickersham
	172878	pSF3-ultraID	Expression of UltraID in mammalian cells with the Tet-On or Tet-Off system	Julien Béthune
	154939	dCas13d-dsRBD-APEX2	RNA-protein interaction mapping via Cas13-based APEX targeting	Alice Ting
	104102–104112	TetBow	Series of plasmids for stochastic multicolor labeling for discriminating between neurons.	Takeshi Imai
	155257	Watermelon Pooled Library	Lentiviral barcode library for simultaneous tracing of clonal lineages and transcriptional and proliferative state of each cell in a population. Proliferation history is monitored by the dilution of a dox-inducible H2B-mCherry marker.	Aviv Regev, Joan Brugge

Content last reviewed 20 June 2025.

References

Das, A. T., Tenenbaum, L., & Berkhout, B. (2016). Tet-On Systems for Doxycycline-inducible Gene Expression. Curr Gene Ther, 16(3), 156–167. https://doi.org/10.2174/1566523216666160524144041 PMID: 27216914

Gossen, M., & Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A, 89(12), 5547–5551. https://doi.org/10.1073/pnas.89.12.5547 PMID: 1319065

Gossen, M., Freundlieb, S., Bender, G., Müller, G., Hillen, W., & Bujard, H. (1995). Transcriptional activation by tetracyclines in mammalian cells. Science, 268(5218), 1766–1769. https://doi.org/10.1126/science.7792603 PMID: 7792603

Heinz, N., Schambach, A., Galla, M., Maetzig, T., Baum, C., Loew, R., & Schiedlmeier, B. (2011). Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum Gene Ther, 22(2), 166–176. https://doi.org/10.1089/hum.2010.099 PMID: 20825282

Krueger, C., Pfleiderer, K., Hillen, W., & Berens, C. (2004). Tetracycline derivatives: alternative effectors for Tet transregulators. BioTechniques, 37(4), 546–550. https://doi.org/10.2144/04374BM04 PMID: 15517964

Loew, R., Heinz, N., Hampf, M., Bujard, H., & Gossen, M. (2010). Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol, 10, 81. https://doi.org/10.1186/1472-6750-10-81 PMID: 21106052

Resnitzky, D., Gossen, M., Bujard, H., & Reed, S. I. (1994). Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol, 14(3), 1669–1679. https://doi.org/10.1128/mcb.14.3.1669-1679.1994 PMID: 8114703

Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., & Hillen, W. (2000). Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A, 97(14), 7963–7968. https://doi.org/10.1073/pnas.130192197 PMID: 10859354

Zhou, X., Vink, M., Klaver, B., Berkhout, B., & Das, A. T. (2006). Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther, 13(19), 1382–1390. https://doi.org/10.1038/sj.gt.3302780 PMID: 16724096

External Resources

• Takara: Tet-inducible Systems Overview
• Tet Systems: Science and Technology