Short hairpin RNA (shRNA)-mediated gene regulation is a commonly used technique for gene manipulation. An efficient and safe delivery system is indispensable when shRNA is delivered into living organisms for gene therapy. Previous studies have proved that DNA and protein can be delivered into dendritic cells (DCs) by non-pathogenic Saccharomyces cerevisiae without being degraded. CD40 is closely related to apoptosis of tumor cells and some immune mechanisms. In this study, we demonstrated that recombinant yeast S. cerevisiae efficiently delivered the shRNA of immune-associated gene (CD40) into mouse intestinal DCs via oral administration. Western blot analysis of isolated intestinal DCs indicated that the inhibition of CD40 gene expression reached up to 56–91%. The secretion of cytokines such as interleukin-2 (IL-2), IL-6, IL-10, IL-12, tumor necrosis factor-α and interferon-γ in intestinal DCs had varying degrees of changes. In conclusion, we found that orally administered recombinant yeast can be used as an efficient shRNA delivery system for intestinal DC-specific gene silencing and immunomodulation in vivo.
Abbreviations: DAS, day after sowing; Gi, germination index; GP, germination percentage; RL, root length; SL, shoot length; SVi, the seed vigor index. Abstract 2Germination of seeds during the transportation or after prolonged storage naturally and inevitably 3 decreases because of ageing, but germination potential can be partially restored with seed priming 4 treatments. A novel attempt was made to investigate the effects of combined treatments and to 5 optimize the conditions for naturally aged seeds of tall fescue (Festuca arundinacea Schreb.), 6 orchardgrass (Dactylis glomerata L.) and Russian wild rye (Psathyrostachys juncea (Fisch.) 7 Nevski) using an orthogonal activity level experimental design [factor A: Azospirillum brasilense 8 concentration, factor B: three seed priming treatments (H 2 O, MgSO 4 and H 2 O 2 ) and factor C: 9 different priming times]. Multivariate regression model analysis was applied to determine the 10 interactive effects of pairwise factors (A and C) and to optimize experimental conditions. The 11results showed that the mixed treatments positively affected seed germination and seedling growth. 12The three seed priming treatments were the dominant factors for germination promotion, whereas 13 the bacterial concentration had the largest effect on seedling growth, especially root elongation.14 The combined results of all determined attributes showed that A. brasilense bio-priming with 15 H 2 O 2 priming constituted the optimal combination. The optimal bacterial concentration of A.16 brasilense and the time of seed priming were 52.3 × 10 6 colony forming units (CFU) mL -1 and 17 17.0 h, respectively. We also discussed the physiological mechanisms that could repair aged seeds:18 the malondialdehyde content and antioxidant enzyme (superoxide dismutase, peroxidase, catalase 19 and ascorbate peroxidase) activities were affected. 20
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