Specificity Of RNA Interference Research Articles

Recipient dendritic cells modified by RNA interference targeting CD80 and CD86 elicit T cell hyporesponsiveness via enhanced T cell apoptosis.

Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood. Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining. There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells. Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

Importance of Translation and Nonnucleolytic Ago Proteins for On-Target RNA Interference

In animals, both siRNAs and miRNAs are thought to diminish protein synthesis from transcripts that are perfectly complementary by directing endonucleolytic cleavage where they anneal, thereby triggering rapid degradation of the entire message [1-4]. By contrast, partially complementary messages are downregulated by a combination of translational repression and accelerated decay caused by rapid poly(A) tail removal [3, 5-12]. Here we present evidence that translational repression can also make a substantial contribution to the downregulation of fully complementary messages by RNA interference. Unlike mRNA destabilization, this inhibitory effect on translation is greater for perfectly complementary elements located in the 3' untranslated region rather than in the protein-coding region. In addition to known disparities in their endonucleolytic activity [13, 14], the four Ago proteins with which siRNAs associate in humans differ significantly in their capacity to direct translational repression. As a result, the relative effect of siRNA on targets that are fully versus partially complementary is influenced by the comparative abundance of the three nonnucleolytic Ago proteins, causing this on-target/off-target ratio to vary in a cell-type-dependent manner because of the dissimilar tissue distribution of these proteins. These findings have important implications for the efficacy and specificity of RNA interference.

Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B: Figure 1.

INTRODUCTIONThis protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage λ (attL1 and attL2). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (attR1 and attR2 flanking a ccdB toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.

Inhibition of VEGF expression by RNA inference (RNAi) technique as a possible new strategy for prevention of human hypertrophic scar

Hypertrophic scarring remains a major problem for patients who have suffered from surgeries or burns. Since the underlying mechanism of hypertrophic scarring is largely unknown, there is no routinely effective treatment for this disease. Vascularization plays an important role in the early phase of hypertrophic scarring. Therefore, the inhibition of angiogenesis may be used as a preventive strategy. Vascular endothelial growth factor (VEGF) has proven to be the most critical angiogenic factor identified to date, which makes this protein an appropriate target for the inhibition of angiogenesis. Therefore, we hypothesize that the anti-angiogenesis by inhibiting VEGF might be used as an effective strategy for early intervention of hypertrophic scar. Due to the high efficiency and specificity of RNA interference (RNAi) technique, we plan to use the vector-based anti-VEGF RNAi system as a powerful, simple and cost-effective tool in future experimental validation. Our hypothesis, as the first description of anti-VEGF by RNAi technique in controlling hypertrophic scar formation is certainly a vital first step for the establishment of an effective anti-scarring modality.

Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi

RNA interference (RNAi) on parasitic nematodes has been described as successful and useful for the identification of novel drug and vaccine candidates. In this study we have evaluated this technology on the cattle parasite Ostertagia ostertagi. Eight different genes were targeted in L1 and L3 O. ostertagi larvae, by electroporation and soaking in dsRNA respectively. Down-regulation of target transcript levels was evaluated by semi-quantitative reverse transcriptase (RT) PCR. In L3 larvae, variable decreases in mRNA levels were observed for 5 genes, ranging from a complete knock down (tropomyosin, beta-tubulin) to a minor decrease (ATPsynthase, superoxide dismutase, polyprotein allergen). However, repeated experiments indicated that effects were sometimes difficult to reproduce. RNAi for ubiquitin, a transthyretin-like protein and a 17 kDa excretion secretion (ES) protein never resulted in a knock down of the transcript. The mRNA levels of 7 non-target genes showed no difference between larvae soaked in C. elegans control dsRNA versus O. ostertagi tropomyosin dsRNA, supporting that the observed reductions are specific for the target gene. Electroporation of L1 larvae proved to be less effective. Reductions in mRNA levels were only noticed for 2 genes and were not reproducible. In conclusion, the results indicate that the RNAi pathway is probably present in O. ostertagi but that the current RNAi techniques can not be used as a reliable screening method.