Background

A crucial technique for studying functional responses in molecular biology is the ability to regulate the expression of a gene. Constitutive gene expression systems have allowed researchers to explore gene responses only if prolonged overexpression is non-toxic. Such systems can also lead to the development of compensatory mechanisms within a cell that can mask functional phenotypes (El-Brolosy et al., 2019). Inducible expression systems are a favored alternative to constitutive expression, giving researchers the ability to switch genes on and off or titrate the level of gene expression, with fewer side effects and greater efficiency. One advantage of such conditional expression is the ability to regulate gene expression in a quantitative, temporally-regulated, and reversible manner that more accurately reveals the direct result of a given genetic change. The tetracycline-controlled operator system containing the TRE promoter provides reversible gene expression with a large induction range at low concentrations of a drug that is non-toxic to mammalian cells. The TRE promoter contains a modified Tet response element consisting of seven repeats of a 36-nt sequence that contains the 19-bp Tet operator sequence (TCCCTATCAGTGATAGAGA). This Tet response element is positioned upstream of a minimal CMV promoter sequence, which lacks the enhancer present in the native CMV promoter. Consequently, the TRE promoter is silent in the absence of binding of the tetracycline responsive rtTA3 regulatory protein. Advances in the system have optimized the reverse Tet transactivator (rtTA) component for improved drug sensitivity and activity and, more importantly, to show no activity in the absence of tetracycline or doxycycline (DOX, a tetracycline analog), thus reducing the leakage of the system (Zhou et al., 2006; Yamada et al., 2018). Nevertheless, it should be noted that tetracycline-derived contaminants are often present in cell culture sera; this poses a challenge for Tet-based systems, but this issue can be avoided by using tetracycline-free serum.

Lentiviruses provide an efficient vehicle for facilitating the establishment of inducible gene expression within a cell, particularly because they are able to infect many different dividing and non-dividing cell types (Naldini et al., 1996; Reiser et al., 1996), making lentiviral systems an essential tool for introducing exogenous genes into difficult to transduce targets such as primary cells. Although lentiviral platforms do pose the risk of endogenous gene disruption, the system described here packages all components into a single vector platform so that only a single viral infection per cell is necessary for tet-inducible gene expression (Figure 1A). This eliminates the need for multiple viral infections and limits the possible number of random genome integrations, thereby limiting the chance of off-target effects on cell physiology (Connolly et al., 2002). The constitutively expressed Neo/Venus transduction marker contained in the system allows for easy enrichment of the transduced cell population and can also be easily replaced with another selection marker (e.g., any antibiotic or cell surface marker) to optimize the system for a range of cell types and applications. This not only makes the system highly tractable, but the development of third generation lentiviral vectors, such as pSLIK (Shin et al., 2006), which separate the minimal genetic elements of HIV into three plasmids (pMDL containing gag and pol, pRSV containing the rev protein, and pVSV containing the envelope protein), increases the safety of lentiviruses and decreases the need for highly specialized training (Barde et al., 2010). While this means that the lentivector system described here requires the transfection of four separate plasmids to generate functional lentiviral particles, this design substantially increases its safety for common laboratory use over second generation platforms that expressed the Gag, Pol, Rev, and Tat genes from a single packaging plasmid. Importantly, third generation lentiviral platforms are self-inactivating in nature due to a deletion in the long terminal repeat (LTR) region that results in the loss of the pro-viral enhancer sequence upon integration. This further improves biosafety because replication-competent viruses are not generated within the target cells (Li et al., 2005). Finally, our lentiviral system utilizes a hybrid 5’LTR fused to a CMV promoter that increases gene expression. This is in contrast to second generation systems, which relied on a weak viral 5’ LTR and required the presence of Tat to activate gene expression. Overall, the third generation lentiviral platform described in this protocol ensures robust gene expression from a single infection of cells and increases the laboratory safety of lentiviruses.

Combining inducible expression with such a flexible lentiviral vector platform allows for tetracycline-regulated gene expression from a minimal viral infection of a wide range of cell targets (Shin et al., 2006). We utilized the Tet-on configuration of such a system in our recent paper (Rommereim et al., 2020) to express Nod-like receptors in THP-1 cells and provide the detailed protocol here.