Tetracycline (Tet) Inducible Expression
To advance the study of gene function, scientists were in search of inducible promoters capable of controlling eukaryotic gene expression. Several endogenous promoters had been identified that responded to stimuli, such as hormones or metal ions; however, these systems were confounded by secondary effects. Scientists began to pursue a non-endogenous system for eukaryotes. At the time, bacterial systems from E. coli presented the best candidates for inducible expression.
Bacterial systems were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested first, and IPTG was found to be inefficient, resulting in low levels of induction. In 1992, Manfred Gossen and Hermann Bujard tested the tet system in a mammalian cell system (HeLa) and found that the tet system was functional, and had rapid induction with efficient tetracycline uptake.
Tet System and the Tet Response Element (TRE)
A TRE is 7 repeats of a 19 nucleotide tetracycline operator (tetO) sequence, and is recognized by the tetracycline repressor (tetR). In the endogenous bacterial system, if tetracycline, or one of its analogs like doxycycline, are present, tetR will bind to tetracycline and not to the TRE, permitting transcription.
Tetracycline-dependent promoters are developed by placing a TRE upstream of a minimal promoter.
Tetracycline Off System
The initial system Gossen and Bujard developed is known as tetracycline off: in the presence of tetracycline, expression from a tet-inducible promoter is reduced. To use tetracycline as a regulator of gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created by fusing tetR with the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex virus).
In the absence of tetracycline, the tetR portion of tTA will bind these tetO sequences and the activation domain promotes expression. In the presence of tetracycline, tetracycline binds to tetR. This precludes tTA binding to the tetO sequences and subsequent increase in expression by the activation domain, resulting in reduced gene expression. This idea of a hybrid transactivator was initially used with the lac system. Tetracycline off is also known as the tTA-dependent system.
Tetracycline On System
In Gossen et al (1995) , random mutagenesis was used to identify which amino acid residues of tetR were important for tetracycline-dependent repression. Mutating these residues led to the development of a reverse Tet repressor, or rTetR, which reversed the phenotype and created a reliance on the presence of tetracycline for induction, rather than repression. The new transactivator rtTA ( reverse tetracycline-controlled transactivator) was created by fusing rTetR with VP16. The tetracycline on system is also known as the rtTA-dependent system.
Choosing a tet system
- If your gene of interest should be active, and only turned off occasionally, using tetracycline off or tTA is more appropriate.
- If your gene of interest should be mostly inactive, and only turned on occasionally, using tetracycline on or rtTA is more appropriate.
- Components for a tetracycline off system are:
- Plasmid containing a tetracycline-dependent promoter upstream of your gene of interest
- tTA expression plasmid
- Components for a tetracycline on system are:
- Plasmid containing a tetracycline-dependent promoter upstream of your gene of interest
- rtTA or TetR expression plasmid
- Stable cell lines can be made that continuously express a system component (e.g. tTA).
Use of tetracycline or a derivative
- A derivative of tetracycline, doxycycline (dox), is a preferred effector for tetracycline transregulation. Dox binds with high affinity to tTA and rtTA; thus, dox can be used in both tetracycline on and off systems.
- Dox also has good tissue distribution, low toxicity, a known half-life (24 hours), and is compararably inexpensive.
- The induction profiles for other tetracycline derivatives can be viewed in Krueger et al, 2004.
- Some cell culture sera may contain tetracycline or its derivatives which can result in unintended system induction. Investigate whether or not your serum is tetracycline-free.
Tetracycline-inducible Empty Backbones
Find a construct that will allow you to insert and induce your gene of interest.
|ID||Plasmid||Description||Co-expressed tTA, rtTA, or TetR||On or Off||PI|
|21916||Tet-pLKO-neo||3rd generation lentiviral plasmid for inducible expression of shRNA; neomycin selection; plasmid 21915 has puromycin selection||TetR||On||Wiederschain|
|41393||pCW57.1||Lentiviral vector for inducible expression for Gateway cloning; selection cassette in format: PGK-rtTA-2A-puro; see article for tagged insert options||rtTA||On||Root|
|11651||pLVUT-tTR-KRAB||Lentiviral vector for inducible expression of transgene or shRNA; see article for detailed information about cloning and additional plasmids||tetR-KRAB||On||Aebischer & Trono|
|16542||pBI-MCS-EGFP||Expression of your gene of interest (MCS with a β-globin poly A) & EGFP from a bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two minimal CMV and is silent in absence of binding of tTA or rtTA||None||On||Vogelstein|
|25735||pSLIK-Neo||Lentiviral vector for inducible expression of a miR-shRNA; selection cassette in format: rtTA3+IRES+Neo; see article for additional selection options||third-generation rtTA||On||Fraser|
|44012||pInducer20||Lentiviral vector for shRNA expression; see article for additional tookit plasmids||third-generation rtTA||On||Elledge|
|19407||pTREtight2||Promoter contains a modified TRE that is silent in absence of binding tTA or rTA; has high copy E. coli origin||None||Either||Ralser|
|64238||pTet-IRES-EGFP||Lentiviral plasmid for inducible expression of transgene of interest and EGFP||None||Either||Lung|
|11662||pPRIME-TET-GFP-FF3||Lentiviral, miRNA expression (PRIME) system for application in knockdown of gene expression at a single copy in mammalian cells; Expresses firefly luciferase hairpin and GFP under pTREtight promoter||None||Either||Elledge|
|35625||pAAV-Ptet-RFP-shR-rtTA||AAV; shRNA cloning vector; for evaluation of shRNA efficacy using fluorescence; use with pGFPns-reporter for cDNA target||rtTA||On||Gu|
|60495||pSBtet-GP||Sleeping Beauty transposon system; has luciferase in cloning site; see article for additional selection and FP option||rtTA||On||Kowarz|
|16623||pBI-GFP||Expression of your gene of interest & GFP from a bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two minimal CMV and is silent in absence of binding of tTA or rtTA||None||Either||Vogelstein|
|100521||pCW57.1-MAT2A||Lentiviral Tet-Off all in one plasmid derived from pCW57.1. rtTA was replaced with tTA, and Puromycin was replaced with Blasticidin selection. Please NOTE, this vector contains an insert (MAT2A) which would need to be replaced by the gene of interest.||tTA||Off||Sabatini|
|92099||AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep||Bidirectional promoter controls expression of gene of interest with Strep-Tag and Tet-On 3G transactivator, creating an auto-regulated Tet-On 3G System. Contains homology arms for integration into AAVS1 Genomic Safe Harbor Locus.||Tet-On 3G||On||Doyon|
|58245||pGLTR-X-GFP||Single vector lentiviral Gateway RNAi system for conditional cell line generation; contains expression cassette for TetR-P2A-GFP; see article for additional constructs||TetR||On||Geley|
Transactivators (tTA or rtTA)
Find a construct that expresses the transactivator for your tetraycline system.
|ID||Plasmid||Description||Element||On or off||PI|
|26429||pLenti CMV rtTA3 Blast (w756-1)||Lentiviral reverse tetracycline-controlled transactivator 3 (rtTA3) expression vector, CMV promoter, and Blasticidin; can be used to make cell lines Tet-On Advanced; plasmid 26730 has hygro resistance||3rd generation rtTA||On||Campeau|
|25434||pMA2640||Retroviral; CMV-driven; linked via IRES to EGFP-Blasticidin fusion; pMA2641 has rtTA driven by retroviral LTR promoters; see article for lentiviral vectors for tet-inducible gene or miRNA expression||rtTA||On||Alexeyev|
|14901||pCL-CTIG||The inducible cassette contains tTA at its 5' end and an inducible promoter at its 3' end, which controls GFP expression||tTA||Off||Verma|
|26803||pEnt L1L3 EF1a-tTA-2||Contains Gateway L1L3 sites and EF1α promoter driving tTA; contains insulator sequence; plasmid pEnt L1L3 rtTA-3 is a rtTA version||tTA||Off||Hsiao|
|34856||fos-tTA||Mouse fos promoter plus part of the first intron (-764/+918) and fos 3'UTR||tTA||Off||Mayford|
|38056||pXCX.CMV.tTA||Adenoviral; CMV promoter; see article for neuronal-specific promoter and enhancer options||tTA||Off||Uney|
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen M & Bujard H. PNAS . 1992 Jun 15;89(12):5547-51.PubMed.
Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Hillen W & Berens C. Annu Rev Microbiol. 1994;48:345-69.PubMed.
Transcriptional activation by tetracyclines in mammalian cells. Gossen M, Freundlieb S, Bender G, Müller G, Hillen W, & Bujard H. Science . 1995 Jun 23;268(5218):1766-9.PubMed.
Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Yao F, Svensjö T, Winkler T, Lu M, Eriksson C, Eriksson E. Hum Gene Ther . 1998 Sep 1;9(13):1939-50.PubMed.
Of mice and models: improved animal models for biomedical research. Bockamp E, Maringer M, Spangenberg C, Fees S, Fraser S, Eshkind L, Oesch F, Zabel B. Physiol Genomics . 2002 Dec 3;11(3):115-32.PubMed.
Tetracycline derivatives: alternative effectors for Tet transregulators. Krueger C, Pfleiderer K, Hillen W, Berens C. Biotechniques . 2004 Oct;37(4):546, 548, 550.PubMed.
Optimization of the Tet-On system for regulated gene expression through viral evolution. Zhou X, Vink M, Klaver B, Berkhout B, Das AT. Gene Ther. 2006 Oct;13(19):1382-90.PubMed.
Improved Tet-responsive promoters with minimized background expression. Loew R, Heinz N, Hampf M, Bujard H, Gossen M. BMC Biotechnol. 2010 Nov 24;10:81. doi: 10.1186/1472-6750-10-81.PubMed.
Tet systems: Science and Technology