InDOXible™ Tet-Activated Lentiviral cDNA Expression System

Background

In 1992, Gossen and Bujard showed that fusion of the bacterial tet-repressor protein with the VP16 activating domain creates a hybrid transactivator protein that binds its target DNA element in a tetracycline-dependent manner. They were able to then use this hybrid tet-regulated transactivator (tTA) to regulate expression from a minimal CMV promoter modified with the tet-responsive tTA target sequence (TRE). This hybrid system provided one of the first, robust technologies for regulating cDNA expression.

Cellecta offers the InDOXible™ Tet-Activated Lentiviral cDNA Expression System, an optimized, tightly regulated inducible gene expression system for virtually any mammalian system. In addition to well-designed vectors, Cellecta has optimized the sequence of the transactivators (i.e., tTA and rtTA) to ensure tightly regulated activation of the TRE Promoter driving expression of the cDNA of interest. Induction in the InDOXible system works optimally with doxocycline, a tetracycline analog. Background levels of antibiotic-selected transduced polyclonal cells cultures is minimal with either the dox-on or dox-off variations of the system.

Some key advantages of the InDOXible Expression System:

• Ultra-low background minimizes leakiness making clonal selection typically unnecessary
• Optional fluorescent sensor enables real-time monitoring of induction and repression
• Versatile design offers easy-to-use all-in-one or two- vector inducible options

Two Complementary Variations

While a number of optimizations have been made to the original tet-inducible cDNA expression system, the basic mechanism remains the same. There are two primary variations of the system:

Dox-Off/tTA: The original design where the VP16 activator is hybridized to the wild-type bacterial transactivator to make a tet-regulated transactivator (tTA), which binds to the TRE unless tetracycline (or the doxycycline analog) is present. This is often referred to as tet-off(or dox-off) functionality, since the addition of a tetracycline analog releases the activator and down-regulates cDNA expression.

Dox-On/rtTA: A variation of the system where the transactivator has been engineered so that it binds only in the presence of a tetracycline analog. It has the reverse activity of the original tTA, and so, is typically denoted rtTA. This variation is referred to as tet-on (or dox-on) functionality, since the additional of tetracycline induces binding to the promoter and expression of the regulated cDNA.

 

Figure 1: All-In-One Lentiviral Dox-On Vector.

The transactivator protein (rtTA or tTA) binds the target TRE Promoter in a tetracycline-dependent manner. The rtTA transactivator binds to the promoter and initiates transcription in the presence of doxocycline (Dox-On), and the tTA transactivator binds and initiates transcription when doxocycline is lacking (Dox-Off).

 All-in-One Lentiviral Dox-On (as shown) and Dox-Off Constructs and Two-Vector TRE-Lentiviral Vector with separate Dox-On and Dox-Off Activator constructs (not shown), are all available.

With the Sensor option, the RFP Sensor is expressed on the same transcript as the cDNA of interest but separated by an IRES (internal ribosome entry site). As a result, it is co-expressed with the cDNA of interest.

 

Two Vector versus All-in-One Vector System:

1. One-Vector Tet-Activated System: The transactivator that regulates gene induction is on the same construct as the inducible gene of interest. Transduction of the single lentiviral construct provides all the elements needed to tightly regulate inducible cDNA expression. 
2. Two-Vector System: The inducible cDNA that is under the control of the responsive promoter is on a vector without the transactivator. The transactivator is on its own vector and transduced separately into cells, and so can be used to create a panel of transactivator cells set up for introduction of different genes of interest.

 

 

Figure 2. Single-Vector Dox-On System Results

U2OS cells were transduced with all-in-one rtTA/TRE-GFP(blastR) reporter vector at MOI<1, then selected with blasticidin for 1 week. Selected cells were cultured for 2 additional days with or without doxycycline, and assayed for GFP fluorescence intensity by flow cytometry. Data shows expression levels of the GFP protein with/without dox.

Note: These are polyclonal cultures. There was no clonal selection. There is very little leakiness.

 

Fluorescent Sensor for Real-Time Induction Monitoring

A fluorescent reporter (RFP) linked by an internal ribosome entry site (IRES) to the cDNA transcript, is available with both the single-vector and dual-vector InDOXible Tet-Activated Vectors. With this IRES configuration, cell fluorescence increases and decreases in parallel with the cloned cDNA allowing monitoring of induction. This fluorescent sensor feature allows convenient confirmation of gene modulation during experiments.

Figure 3. Dox-On System with RFP Sensor

U2OS cells were transduced (MOI <1) with the TRE-GFP-IRES-RFP reporter vector, selected with blasticidin for 1 week, and cultured for 2 additional days with or without doxycycline. Detection levels for both GFP and RFP fluorescence intensity were assessed by flow cytometry. Activation of the RFP sensor signal correlates strongly with the increased activation of the GFP cDNA of interest.

 

References:

 Gossen M., Bujard H., Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51.