The Basic System: Components and Principles
The Tet Technology comprises two complementary control circuits, initially
described as the tTA dependent [1] and rtTA dependent [2] expression systems.
They are now commonly referred to as the Tet-Off System (i. e. tTA dependent)
and the Tet-On System (i. e. rtTA dependent).
In each system, a synthetic tetracycline controlled transcription factor (tTA
or rtTA) interacts with a tTA/rtTA responsive promoter, Ptet, to drive expression
of a gene under study. Expression is regulated from outside by the effector
substance tetracycline or one of its derivatives. Tetracyclines act at the
level of DNA binding of tTA and rtTA transcription factors. rtTA requires a
tetracycline ligand for DNA binding, whereas the interaction between tTA and
DNA is prevented by tetracycline. Thus, the two versions of the Tet System
respond to tetracyclines in opposite manner, making them complementary as each
system has its unique characteristics and strengths.
High induced expression levels combined with low basal background result in
the excellent dynamic range of both expression systems, allowing regulation
of gene expression over many orders of magnitude without interference with
host cell physiology.
The Tet System owes these characteristics
to |
|
| - | the prokaryotic origin of its core components, the Tet repressor, TetR,
and its cognate binding site, the tet operator, tetO, making the system
essentially inert in an eukaryotic environment; |
| - | the pharmacological properties of its effectors, in particular doxycycline
(Dox), an exhaustively characterized antibiotic widely applied in man and
animals; |
| - | the favourable thermodynamic parameters of both the repressor/operator
as well as repressor/effector interaction. |
Successful optimizations and adjustments of the components constituting the regulatory circuits resulted in expression systems that can be readily set up "as is“ (following a standard procedure), or conveniently adapted to special requirements thanks to the modular nature and compatibility of their elements.
As depicted in Figure 1, the two Tet dependent control circuits
consist of four basic elements: |
|
| • | tTA is a hybrid transcription factor resulting from the fusion
of the prokaryotic Tet repressor, TetR, with a eukaryotic transcriptional
transactivation domain (most widely used so far is the acidic domain of
HSV VP16). The TetR moiety confers sequence specific DNA binding, sensitivity
to tetracyclines and dimerization to the tTA fusion protein. Accordingly,
the response of both the TetR and tTA to tetracyclines is similar: binding
of the antibiotic dramatically lowers their affinity to their common cognate
binding sites, the tet operators. |
| • | rtTA differs from tTA by a few point mutations within
TetR. These, however, result in a complete reversal of tetracycline responsiveness
of the
transcription factor. rtTA requires tetracyclines for binding to tetO.
Note that specific tetracycline derivatives like Dox or anhydrotetracycline
(ATc) have to be used to fully reveal the rtTA phenotype. |
| • | Ptet is a synthetic promoter responsive to both tTA and rtTA. It comprises
a minimal RNA polymerase II promoter (transcriptionally silent in the absence
of additional transcription factor binding sites) fused to multimerized
tetO sequences (Fig.2). This arrangement makes the activity of
Ptet dependent on the binding of tTA or rtTA. The design of such synthetic
tTA/rtTA responsive
promoters is flexible with respect to both the origin of the minimal promoter
as well as the exact arrangement of the operators. The original version
which consists of a CMV minimal promoter fused to an array of seven tetO sequences is designated Ptet-1. It is commercially distributed as part
of the pTRE vector series (for tetracycline responsive element), somewhat
in line with the prevailing eukaryotic nomenclature. |
| • | Doxycycline, a tetracycline derivative, is currently the most suitable
effector substance for both the Tet-On and the Tet-Off System. It binds with high affinity to tTA as well as to rtTA and, thus, is fully effective in the Tet-Off system at such low concentrations as 1-2 ng/ml in the case of tTA and in the Tet-On systems effective down to concentrations as low as 80 ng/ml in the case of rtTA2-syn1. An excellent medical safety record and well characterized pharmacological properties like superb tissue penetration and low toxicity in eukaryotes make Dox the effector substance of choice for most applications in tissue cultures and whole organisms. |