Lentivirus-mediated Conditional Gene Expression

Abstract

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells. This is done in a third generation lentiviral expression platform that improves the safety of lentiviruses and allows for greater gene expression than previous lentiviral platforms. The lentiviral expression plasmid is first engineered to contain the gene of interest driven by a TRE (tetracycline response element) promoter in a simple gateway cloning step and is then co-transfected into HEK293T cells, along with packaging and envelope plasmids to generate the virus. The virus is used to infect a cell type of interest at a low MOI so that the majority of the transduced cells contain a single viral integration. Infected cells are grown under selection, and viral integration is validated by qPCR. Gene expression in stably transduced cells is induced with doxycycline and validated by qPCR, immunoblot, and flow cytometry. This flexible lentiviral expression platform may be used for stable and robust induction of a gene of interest in a range of cells for multiple applications. Graphic abstract: Schematic overview of lentiviral transduction of THP-1 cells.

Keywords: Lentivirus; NLR; NOD1; Stable expression; TRE; Tetracycline.

Figure 1.. Lentivirus-mediated conditional expression of NOD1 and NLRP2.

(A) Lentiviral system for DOX-inducible expression of NLRs. Expression of 3X-FLAG-tagged NLR coding sequence was transcribed under the control of an inducible tetracycline promoter, called a tetracycline response element (TRE). To engineer the lentiviral expression vectors pSLIK_Neo_NOD1 or pSLIK_Neo_NLRP2, N-terminal 3X-FLAG-tagged NOD1 or NLRP2 were first cloned between the Spe1 and Mfe1 restriction sites of the entry vector pEN_TmiRc3, so that their gene expression was driven by a TRE promoter. Entry vectors were then recombined with the lentiviral expression vector, pSLIK_Neo, via the Gateway LR cloning reaction. This recombination reaction shuttles the TRE-driven NOD1 and NLRP2 gene cassettes into the lentiviral backbone, resulting in the generation of pSLIK_Neo_NOD1 and pSLIK_Neo_NLRP2 lentiviral expression vectors. Lentiviral plasmids were co-transfected with pMDL, pRSV, and pVSV into HEK293T cells to generate lentiviruses used to infect THP-1 cells. (B) qPCR showing construct:chromosome ratio of the conditionally expressed NLRs. THP-1 cells were infected with lentivirus at low infectivity. qPCR probes specific for the endogenous or the FLAG-tagged NLR (Table 1) were used to determine the ratio of the NLR transgene (construct) to the endogenous gene (chromosome), as an estimate of the number of integrations per genome. A construct:chromosome ratio of 0.5 indicates one integration per cell, and a ratio of 1 indicates two integrations per cell. (C) qPCR showing overexpression of NLRs upon DOX addition. THP-1 cells harboring DOX-inducible NOD1 or NLRP2 were left untreated or were treated with DOX for 6 h. RNA was harvested, and NLR expression was determined by qPCR. NLR expression in the absence of DOX was set at 1. DOX-induced NLR overexpression was measured relative to that in the absence of DOX. (D and E) Immunoblot (D) and flow cytometry (E) showing protein expression of FLAG-tagged NLRs in the indicated THP-1 lines with or without addition of DOX for 6 h. ‘Vector’ refers to cells transduced with empty vector and ‘control’ refers to THP-1 cells with no lentiviral transduction. Data are presented as means ± SEM. Statistical analyses were performed using a Student’s unpaired t-test (two-tailed) in GraphPad Prism. P values <0.05 were considered significant. ****P < 0.0001.