Browsing by Subject "Transfection"

Now showing 1 - 5 of 5
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    Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo
    (Springer Nature, 2021) Xuan, Yi; Ghatak, Subhadip; Clark, Andrew; Li, Zhigang; Khanna, Savita; Pak, Dongmin; Agarwal, Mangilal; Roy, Sashwati; Duda, Peter; Sen, Chandan K.; Surgery, School of Medicine
    Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.
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    Inhibition of Small-Conductance, Ca2+-Activated K+ Current by Ondansetron
    (Frontiers Media, 2021-04-22) Guo, Shuai; Chen, Zhenhui; Chen, Peng-Sheng; Rubart, Michael; Medicine, School of Medicine
    Background: Small-conductance Ca2+-activated K+ channels (SK channels) have been proposed as antiarrhythmic targets for the treatment of atrial fibrillation. We previously demonstrated that the 5-HT3 receptor antagonist ondansetron inhibits heterologously expressed, human SK2 (hSK2) currents as well as native cardiac SK currents in a physiological extra-/intracellular [K+] gradient at therapeutic (i.e., sub-micromolar) concentrations. A recent study, using symmetrical [K+] conditions, challenged this result. The goal of the present study was to revisit the inhibitory effect of ondansetron on hSK2-mediated currents in symmetrical [K+] conditions. Experimental Approach: The whole-cell patch clamp technique was used to investigate the effects of ondansetron and apamin on hSK2-mediated currents expressed in HEK 293 cells. Currents were measured in symmetrical [K+] conditions in the presence of 100 nM [Ca2+]o. Results: Expression of hSK2 produced inwardly rectifying whole-cell currents in the presence of 400 nM free cytosolic Ca2+. Ondansetron inhibited whole-cell hSK2 currents with IC 50 values of 154 and 113 nM at -80 and 40 mV, respectively. Macroscopic current inhibited by ondansetron and current inhibited by apamin exhibited inwardly rectifying current-voltage relationships with similar reversal potentials (apamin, ∼5 mV and ondansetron, ∼2 mV). Ondansetron (1 μM) in the continuing presence of apamin (100 nM) had no effect on hSK2-mediated whole-cell currents. Wild-type HEK 293 cells did not express ondansetron- or apamin-sensitive currents. Conclusion: Ondansetron in sub-micromolar concentrations inhibits hSK2 currents even under altered ionic conditions.
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    Massively-Parallelized, Deterministic Mechanoporation for Intracellular Delivery
    (American Chemical Society, 2020-02) Dixit, Harish G.; Starr, Renate; Dundon, Morgan L.; Pairs, Pranee I.; Yang, Xin; Zhang, Yanyan; Nampe, Daniel; Ballas, Christopher B.; Tsutsui, Hideaki; Forman, Stephen J.; Brown, Christine E.; Rao, Masaru P.; Medicine, School of Medicine
    Microfluidic intracellular delivery approaches based on plasma membrane poration have shown promise for addressing the limitations of conventional cellular engineering techniques in a wide range of applications in biology and medicine. However, the inherent stochasticity of the poration process in many of these approaches often results in a trade-off between delivery efficiency and cellular viability, thus potentially limiting their utility. Herein, we present a novel microfluidic device concept that mitigates this trade-off by providing opportunity for deterministic mechanoporation (DMP) of cells en masse. This is achieved by the impingement of each cell upon a single needle-like penetrator during aspiration-based capture, followed by diffusive influx of exogenous cargo through the resulting membrane pore, once the cells are released by reversal of flow. Massive parallelization enables high throughput operation, while single-site poration allows for delivery of small and large-molecule cargos in difficult-to-transfect cells with efficiencies and viabilities that exceed both conventional and emerging transfection techniques. As such, DMP shows promise for advancing cellular engineering practice in general and engineered cell product manufacturing in particular.
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    MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU
    (Public Library of Science, 2010-04-26) Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Neves, Ricardo Pires das; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L.; Medicine, School of Medicine
    Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. Methods and Findings In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Conclusions Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.
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    Molecular tools for analysis of gene function in parasitic microorganisms
    (Springer, 2007) Meissner, Markus; Agop-Nersesian, Carolina; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    With the completion of several genome sequences for parasitic protozoa, research in molecular parasitology entered the "post-genomic" era. Accompanied by global transcriptome and proteome analysis, huge datasets have been generated that have added many novel candidates to the list of drug and vaccine targets. The challenge is now to validate these factors and to bring science back to the bench to perform a detailed characterization. In some parasites, like Trypanosoma brucei, high-throughput genetic screens have been established using RNA interference [for a detailed review, see Motyka and Englund (2004)]. In most protozoan parasites, however, more time-consuming approaches have to be employed to identify and characterize the function of promising candidates in detail. This review aims to summarize the status of molecular genetic tools available for a variety of protozoan pathogens and discuss how they can be implemented to advance our understanding of parasite biology.