Pre-made lentiviral expression particles (lentivirus) can be used for transient or stable gene expression in dividing and non-dividing cells without the use of transfection reagents. Some of GenTarget’s lentiviral particles are generated from its optional inducible lentivectors.
What is GenTarget’s optional inducible expression?
Constitutive expression or knockdown of certain genes may be toxic or unwanted; in such cases, controlled expression is required. GenTarget’s optional inducible lentiviral particles address this need by this need by allowing either constitutive or inducible gene expression in the same lentivirus. Target or shRNA are expressed under a modified suCMV promoter or H1 promoter, respectively. Both promoters contain two copies of the tetracycline operator sequence. This modification does not silence the promoters’ constitutive expression; however, in the presence of a repressor protein (TetR), the promoters are repressed by the binding of TetR to the Tet operator (TetO) sequence. Once tetracycline (Tet) or its derivative Doxycycline (Dox) is added, it binds the TetR protein, which is then released from the promoter, allowing expression / knockdown to proceed.
Inducible expression is optional. When no TetR is present, the promoter functions as a strong constitutive promoter without any need for induction. When inducible expression is desired, introduction of TetR in advance stops expression, which can then be induced by adding tetracycline or Dox. (See the induction mechanism figure below).
How to deliver the repressor regulator (TetR) for inducible expression?
The repressor protein (TetR) must be present in order to use the particles for Tet-inducible expression. The presence of tetR can be achieved by the following methods:
- Pre-made TetR expression lentivirus: This is the most effective and easiest method to deliver TetR expression. Simply add GenTarget’s TetR expression lentivirus and select the tetR expressing cells for downstream application.
- Transfection of a TetR expression plasmid: Transfect the tetR expression plasmid in advance, or co-transduce both the tetR repressor lentivirus and the target inducible expression lentivirus into the sample cell line.
How much tetracycline (or Dox) is used for induction? How much induction can be expected?
Our inducible system has a low basal expression level and very high fold induction. Expression is induced in the presence of tetracycline or doxycycline in a dose-dependent manner. The amount of tetracycline required is dependent upon cell type, but a final concentration of 1.0 µg/ml is commonly use. Please note that many bovine sera used in culture are contaminated with tetracycline or its derivatives, which can affect basal expression. Depending upon the cell line and the repressor protein expression level, a 20-fold to 1000-fold increase in expression can normally be obtained after induction.
The following picture demonstrates GFP expression from premade inducible GFP lentiviral particles (Cat#: LVP024) in a repressor stable cells (CAT#: SC005-Bsd) before and after induction.
What’s the difference between our inducible system and the Tet-on / Tet-Off system? Are they compatible?
Our lentiviral inducible expression vector contains a strong constitutive promoter (H1 or CMV) integrated with two copies of the tetracycline regulator binding sequence. The shRNA or the gene of interest (GOI) can be expressed at a high level without any induction or activation. Optionally, expression can be first repressed in the presence of the repressor protein (TetR) which binds to the expression vector’s Tetracycline Response Element (TRE), and then induced by addition of tetracycline (which removes TetR from the TRE).
The expression vector for the Tet-On/Tet-Off systems contains a silent promoter and uses a modified form of TetR that functions as a transcriptional activator rather than a transcriptional repressor; expression is turned on only after this transcriptional activator binds to the TRE. The binding of the transcriptional activator to the TRE is dependent upon the presence (Tet-on), or absence (Tet-off) of tetracycline or doxycycline. Our system may not fully compatible with the TetOn/Off system since they are different systems.
Please click to see our optional inducible expression lentivirus for human, mouse and rat genes.
References:
- O’ Gorman et al., 1991; Sauer, 1994.
- Molecular Therapy (2003) 7, 460-466;
- Ann. Rev Microbiol. 1994; 48:345-69.
- Microbiol Mol Biol Rev. 2005 Jun; 69(2):326-56.