Tetracycline-Inducible Systems and Vectors

Cellecta offers a range of inducible lentiviral vectors that enable modulation of both short RNA sequences (e.g., shRNA and sgRNA), and standard mRNA transcripts.

  • Two tet-inducible systems for both cDNA and short RNA (e.g., sgRNA, shRNA) expression
  • Tightly regulated doxycycline-responsive promoters
  • All-in-one, single vector designs that include both the promoter and tet-responsive regulator
  • Cellecta provides inducible expression systems for both short RNA sequences, such as sgRNA and shRNA, using an expression cassette designed for RNA polymerase 3 with tet-responsive U6 or H1 promoters, as well as for inducible cDNA gene expression with the InDOXible tet-responsive RNA polymerase 2 promoter. The two distinct tet-inducible systems enable regulated expression of almost any genetic sequence.

    Inducible sgRNA and shRNA: The inducible U6 (or H1) promoter driving sgRNA/shRNA expression in our constructs is driven by RNA polymerase 3, which transcribes short RNA expression, like rRNA, and tRNA. We developed the tet-inducible U6 promoter in-house at Cellecta.

    Expression of cDNA:Most researchers are more familiar with the widely used tet-inducible expression system that makes use of a modified CMV promoter to drive transcription of protein-encoding mRNA. Cellecta has also developed an optimized variation of this Tet-Activated system, which is regulated by the rtTA activator or tTA repressor.

    These are two distinct systems that provide regulatable expression for different genetic sequences.

    Cellecta offers all the tet-inducible vector options as part of its sgRNA, shRNA, and cDNA custom lentiviral vector construction services, and also sells the individual vectors separately for researchers interested in making their own constructs.

  • Cellecta offers inducible tet systems for both RNA polymerase 3 expression of sgRNA and shRNA, and for RNA polymerase 2 expression of cDNA transcripts.

    (A) Inducible sgRNA and short hairpin sequences:Expression of sgRNA and shRNA is driven by the human U6 promoter in most mammalian vectors. RNA polymerase 3 binds to U6, H1 and other similar promoters to drive expression of short RNA sequences (e.g., rRNA, tRNA, etc.). Cellecta has developed an inducible version of the U6 promoter by combining it control elements from the bacterial tet-operator into the U6 promoter. When the Tet-Repressor (TetR gene) binds to the modified U6 promoter, transcription is blocked. If tetracycline (or doxycycline) is present, the Tet-Repressor cannot bind, and transcription is active. Almost all our inducible sgRNA and shRNA vectors are available as all-in-one systems, in that, they contain both the sgRNA/shRNA expression cassette and the TetR gene that regulates expression.

    (B) Inducible cDNA Expression: Cellecta’s Tet-Responsive InDOXible™ System regulates expression of cDNA. This system is built around a hybrid activator protein (i.e., tTA for Dox-Off) or repressor protein (i.e., rtTA for Dox-On) with a modified CMV promoter (PTRE), which is driven by RNA polymerase 2. Cellecta offers vectors which have both the rtTA or tTA regulator in the same construct as the tet-responsive promoter, and versions where the regulator protein is expressed on a separate construct. Depending on the application, it may be preferable to make a cell line that expresses the tTA or rTA regulator protein, then use it to make a panel of inducible cDNA expression lines.

  • There are numerous applications where inducible expression of genes or effectors can help address key experimental questions or more rapidly advance research findings. Below are some common uses:

    • Inducible expression of proteins  to evaluate gene function
    • Up- and down-regulation of a target gene or effector for a specific gene to induce tumorigenesis, activate a pathway, induce differentiation, or stimulate some other phenotype
    • Suppress expression of toxic gene until required for an experiment.
    • Reversibly regulate activation or repression of a target gene CRISPRa, CRISPRi, or shRNA.
    • Assess whether a particular gene is required for a biological response to a drug or compound.
    • Monitor Cas9 induction in real time with Cas9-BFP