Tetracycline-inducible shRNA systems allow you to tightly regulate the expression of functional short hairpin RNAs (shRNAs) in mammalian cells in order to silence target genes and are especially useful in cases where suppression of a gene may be lethal.
There are three versions of Clontech's tetracycline-inducible shRNA technology:
- The Knockout Single Vector Inducible RNAi System. Our most convenient system. Once your shRNA sequence is cloned in this vector, the single plasmid contains all the features necessary for inducible gene knockdown. Using this system can save weeks of time because your inducible shRNA-expressing stable cell line is produced after only a single round of transfection and selection.
- The Knockout Tet RNAi Systems H and P. These hygromycin (H)- and puromycin (P)-selectable systems feature inducible retroviral shRNA expression vectors, which are provided prelinearized and ready for ligation with a dsDNA oligonucleotide encoding your shRNA. The procedure is a 2-step process of sequential retroviral transductions. First, a Tet-responsive cell line is created by transducing, selecting, and screening stable cell clones expressing the system’s regulatory protein, the tTS transcriptional silencer. Next, your tTS cell clone (or a tTS Cell Line available from Clontech) is transduced by your shRNA expression retrovirus, then selected and screened for clones that inducibly express your shRNA in the presence of doxycycline (Dox).
The Inducible RNAi Mechanism
The system is designed so that expression of an shRNA is induced when doxycycline (Dox; a tetracycline analog) is added to the culture medium.
The system relies on two components:
- the tTS regulatory protein, which is a tetracycline-controlled transcriptional silencer, and
- a tetracycline inducible promoter (PTightU6), the activity of which is regulated by tTS binding.
The tTS protein is a fusion of the Tet repressor protein (TetR) and a KRAB silencing domain, a powerful transcriptional suppressor. In the absence of Dox, tTS tightly binds Tet operator sequences (tetO
) located in PTightU6
and acts as a potent suppressor of transcription.
Fast Response Times & High Sensitivity
With all of our Inducible RNAi systems, knockdown of your target gene’s expression can be detected within 24 hr of Dox addition, and maximum knockdown is typically seen within 48 hr. This rapid response is possible because transcription from PTightU6 is actively suppressed by tTS, rather than it being merely repressed by simple steric hindrance.
In contrast, other inducible expression systems relying on steric inhibition exhibit slow induction (up to several days) and may require pretreatment with Dox for 1–2 days prior to transfection to ensure that repression is fully alleviated. This can result in incomplete induction of shRNA (compared to repressor-free controls). Also, high levels of Dox (2–5 µg/ml) are often needed to ensure full displacement of the repressor, which may have deleterious effects on other cell functions. Our systems are sensitive to very low, nontoxic Dox concentrations (67% knockdown at 1 ng/ml).