pCCL-PGK-eGFP-4 Tough Decoy-203 was cloned analogously by sequential cloning of the Tough Decoy-203 sequence, PCR-amplified with primers introducing additional restriction sites for sequential insertion of the four Tough Decoy sequences. activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed DNQX in human disease. = 1.6 10?7) and miR-203 (= 1.1 10?7), respectively. To further validate this mutual ranking of the three most potent inhibitors, we performed a titration assay in which increasing dosages of the three inhibitor-encoding plasmids were transfected into HEK-293 cells and observed a clear dose-response correlation for all inhibitors (data not shown). At all concentrations of inhibitor-encoding plasmid, Tough Decoy inhibitors performed better than both Sponges, and Bulged Sponges performed marginally better than Sponges DNQX with perfect miRNA complementarity (data not shown). Rabbit Polyclonal to SRY When the inhibitors were expressed from transduced lentiviral vectors, we observed that only vectors encoding Tough Decoy and Bulged Sponge inhibitors were able to suppress the activity of the target miRNAs with statistical significance relative to the negative control (Fig. 2C,D). Notably, we found for both miR-16 and miR-203 that lentiviral vectors encoding Tough Decoy inhibitors resulted in levels of RLuc expression that were significantly higher than those obtained by lentiviral transfer of Bulged Sponge inhibitors (= 0.018 and = 0.033 for miR-16 and miR-203, respectively). Together, these experiments demonstrate that, among seven different types of miRNA inhibitors, the most potent interference of miRNA activity was obtained by Tough Decoy inhibitors expressed from both plasmid DNA and lentiviral vectors. Open in a separate window FIGURE 2. Tough Decoy inhibitors perform best among seven miRNA inhibition strategies when delivered by plasmid transfection or lentiviral transduction. A dual-luciferase assay was used to screen the potency of seven vector-encoded miRNA inhibitors targeting miR-16 (< 0.05, (**) < 0.01, (***) < 0.001, (****) < 0.0001. Variable transductional titers of inhibitor-encoding lentiviral vectors The transduction efficiency of lentiviral vectors encoding miRNA inhibitors is potentially affected by the presence of an inhibitor cassette which may disturb the performance of the vector in both producer and recipient cells. Not only could complex secondary structures of the inhibitor affect transcription, reverse transcription, and packaging of the viral genome, but lentiviral vector RNA is also potentially subjected to degradation due to recognition of the inhibitor sequence by the complementary miRNA. Also, it cannot be excluded that miRNA inhibition may affect the virus-producing cells or that the presence of the inhibitor expression cassette in the 3 LTR may have a negative impact on virus production since inserts within the 3 LTR reduce viral titers proportionally to the length of the insert (Urbinati et al. 2009). To address the impact of the different inhibitors on vector transfer, we first determined transductional titers as measured by the number of puromycin-resistant colony-forming units obtained in vector-transduced HeLa cells (Fig. 3A). Marked variations in titers, ranging from 2 107 CFU/mL for LV/Mask-16 to 2 104 CFU/mL for LV/Sponge-16, were observed among the miR-16 inhibitor-encoding lentiviral vectors, whereas titers for all miR-203-inhibiting vectors were high and did not vary significantly (titers ranging from 1 107 to 4 107 CFU/mL). Together with the fact that miR-16 was highly DNQX expressed and miR-203 was only vaguely expressed in virus producer and recipient cells (HEK-293T and HeLa cells, respectively) (Fig. 1C), these data demonstrated that vector transduction for some of the inhibitors was strongly influenced by endogenous miRNAs. Notably, we found that the titers among the two most potent inhibitor-encoding vectors, LV/Tough Decoy-16 DNQX and LV/Bulged Sponge-16, varied 100-fold. Such substantial titer variations between miR16-targeting vectors were potentially caused by the design of the lentiviral vector (Fig. 1B) in which the presence of only a single polyadenylation signal in the 3 LTR rendered the puromycin N-acetyl-transferase (PAC) mRNA transcript susceptible to miRNA-mediated regulation due to the miRNA target sites in the inhibitor. Hence, these data suggest that vectors encoding a classical Sponge configuration were highly vulnerable to endogenous miRNAs, whereas Tough Decoy-containing transcripts were less sensitive to targeting by their complementary miRNA. Open in a separate window.
