A multifunctional lentiviral-based gene knockdown with concurrent rescue that controls for off-target effects of RNAi

Abstract

The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 (α-ACTNl) expression in human T cells. α-ACTNl RNAi resulted in inhibited chemotaxis to SDF-lα, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTNl (rr-α-ACTNl) in the same cell. The presence of a GFP tag on rr-α-ACTNl allowed for detection of appropriate subcellular localization of rr-α-ACTNl. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.

Figure 1

Multifunctional lentiviral vectors. A. Map of the lentivrial vectors. pFLRu was generated as described in Materials and Methods. There are two MCSs: 5’ of UbiC has the MCS for shRNA expression cassette, and 3’ of Ubic has the MCS for RNAi-resistant isoform of shRNA targeted gene containing an in-frame C-terminal GFP tag and FH. It also contains a puromycin (puro) resistance cassette. B. Jurkat T cells were infected with freshly produced lentivirus containing pFLRu-α-ACTN1 shRNA, puromycin selected, and FACS analysis performed. The percentages of GFP positive cells are presented. Left panel: parental Jurkat T cells; right panel: lentiviral transduced Jurkat T cells.

Figure 2

Lentiviral-based Dicer-dependent α-ACTN1 shRNA worked more potent than Dicer-independent counter part. A. Putative 27-mer shRNA for α-ACTN1 and predicted shRNA cleavage sites are indicated in arrows. B. Putative 20-mer shRNA for α-ACTN1. C. Western blot analysis of Jurkat T cells transduced with lentivirus expressing control shRNA (lane 1), α-ACTN1 27-mer shRNA (lane 2), and α-ACTN1 20-mer shRNA (lane 3). Jurkat T cells were transduced by viruses carrying corresponding shRNA expression cassette, and selected by puromycin. The residue cells were collected, and the cell lysate was prepared for Western blots using anti-α-ACTN1 antibody as primary antibody.

Figure 3

Cellular and functional characterization of multifunctional lentivirus. A. Mutations in RNAi-targeted sequence of human α-ACTN1 (red) to generate rr-α-ACTN1. B. Western blot analysis. α-ACTN1 levels in Jurkat T cells transduced with control shRNA lentivirus (lane 1), α-ACTN1 shRNA lentivirus (lane 2), and α-ACTN1 shRNA/rr-α-ACTN1-GFP (lane 3) were detected using anti-α-ACTN1 antibody. The α-tubulin levels were detected as a loading control using anti α-tubulin antibody. 30 μg of protein from each cell lysate was applied in each lane. C. Subcellular localization of endogenous α-ACTN1 and rr-α-ACTN1-GFP in Jurkat T cells transduced with control shRNA lentivirus (i-iii), α-ACTN1 shRNA lentivirus (iv-vi), and α-ACTN1 shRNA/rr-α-ACTN1-GFP lentivirus (vii-ix). The lower panel (x-xii) demonstrates co-localization of rr-α-ACTN1-GFP and F-actin. Cells were stained with the polyclonal antibody against GFP (i, iv, vii and x), the monoclonal antibody against α-ACTN1 (ii, v and viii), or Rhodamine phalloidin (xi). Confocal images were obtained. D. α-ACTN1 shRNA depletion of endogenous α-ACTN1 in Jurkat T cells results in inhibited chemotaxis to SDF-1α that is rescued by co-expression of rr-α-ACTN1-GFP. Values represent average +/− standard deviation of triplicates. The experiments were performed three separate times with similar results.