Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown

Abstract

Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery.

Figure 10.1

Knockdown of ASC in THP1 cells transduced using lentiviral shRNA vector, FG12 (14). THP1 cells were transduced with lentivirus expressing shRNA against ASC. Our previous studies have shown efficacy for these same shRNAs in reducing ASC expression when expressed using a stem cell virus-based retroviral vector pHSPG (6, 13). Similar to our previous results, the shASCs transduced using FG12 reduce endogenous ASC levels in THP1 monocytic cells by approximately 80% (shASC#1) and 60% (shASC#2). Three control cell lines were also tested for comparison, untransfected THP1 cells (THP1), cells transduced with an empty lentiviral vector (EV), and cells transduced with a lentivirus expressing a scrambled target for shASC#1 (mut-shASC#1). Results represent the averages plus standard deviations of triplicates, are standardized to 18s rRNA expression, and are normalized to an average of 100 in THP1 cells.

Figure 10.2

ELISA of IL-1β in control and shASC knockdown cell lines following infection with 10 MOI Porphyromonas gingivalis. This figure demonstrates how a functional assay can be used to verify knockdowns. In this case of our protein of interest, ASC, has a well-established role in processing IL-1β following infection with bacteria (10, 11, 13). The reduced IL-1β that is observed for the shRNA cell lines following infection with P. gingivalis verifies the knockdowns. Additionally, the experiment shows dose dependency since the shASC#2 is less effective than shASC#1 in knocking down ASC (Fig. 10.1) and also has proportionally less efficacy in reducing IL-1β secretion levels. This figure, therefore, illustrates both the general utility of a functional assay and the advantage of having two different knockdowns of different efficacy to verify dose-dependent functional effects. The use of two shRNAs also provides an additional level of control for studies of ASC function since two shRNAs are statistically unlikely to promote the same off-target knockdowns.