Adenovirus-mediated gene transfer.
ABSTRACT Recombinant adenoviruses are attractive vectors for short-term expression in mouse liver and primary cell lines. Various versatile vector systems have been developed which can be used for the reliable production of recombinant adenoviruses. This protocol describes the entire process for the production of recombinant adenoviruses using the AdEasy system. This protocol will give a practical step-by-step description from the cloning of the gene of interest until the in vivo administration in mice. The entire process will take about 8 weeks to complete.
SourceAvailable from: Cheng-Deng Kuo[Show abstract] [Hide abstract]
ABSTRACT: Activation of Toll-like receptor 4 (TLR4) triggers both innate and adaptive immunity. We previously identified a synthetic glycolipid, CCL-34, which can induce anticancer immunity in a TLR4-dependent manner. In the present study, we demonstrated the involvement of THO complex 1 (thoc1) in the CCL-34-induced anticancer mechanism. The expression of thoc1 was suppressed in bladder cancer cells (MBT-2) co-cultured with CCL-34-activated macrophages, whereas treatment with an iNOS inhibitor could restore the expression of thoc1. Direct treatment of MBT-2 cells with an NO donor also repressed thoc1 expression. Importantly, the thoc1-overexpressing MBT-2 cells (MBT/thoc1) exhibited greater resistance than the MBT-2 cells to cytotoxicity induced by the NO donor or the CCL-34-activated macrophages. In addition, treatments with CCL-34-activated macrophages or the NO donor resulted in the suppression of thoc1 promoter activity in MBT-2 cells, and mutations in the antioxidant response element (ARE) of the thoc1 promoter abolished the repression induced by these treatments. Furthermore, NO treatment increased the expression and nuclear localization of nuclear factor E2-related factor 2 (Nrf2) in MBT-2 cells. Overexpression of Nrf2 suppressed thoc1 promoter activity in an ARE-dependent manner, and knock-down of nrf2 reversed the suppression. Notably, Bcl-2 expression was suppressed in MBT-2 cells, but not in MBT-2/thoc1 cells, treated with CCL-34-activated macrophages or the NO donor. In summary, our results demonstrate that NO-mediated thoc1 downregulation, via Nrf2, is a key step in the cancer cell apoptosis induced by CCL-34-treated macrophages and that downregulated thoc1 could lead to Bcl-2 downregulation and subsequent cancer cell apoptosis.Biochemical pharmacology 05/2013; DOI:10.1016/j.bcp.2013.05.009 · 4.65 Impact Factor
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ABSTRACT: Protons activate ASIC1a in the central nervous system (CNS), though the impact of such activation on brain outputs remains elusive. Progress elucidating the functional roles of ASIC1a in the CNS has been hinder by technical difficulties of achieving acidification with spatial and temporal precision. We have implemented a method to control optically the opening of ASIC1a in brain slices and also in awake animals. The light-driven H+ pump ArchT was expressed in astrocytes of mouse cortex by injection of adenoviral vectors containing a strong and astrocyte-specific promoter. Illumination with amber light acidified the surrounding interstitium and led to activation of endogenous ASIC1a channels and firing of action potentials in neurons localized in close proximity to ArchT-expressing astrocytes. We conclude that this optogenetic method offers a minimally invasive approach that enables examining the biological consequences of ASIC1a currents in any structure of the CNS and in the modulation of animal behaviors.Journal of Biological Chemistry 04/2014; 289(22). DOI:10.1074/jbc.M114.550012 · 4.60 Impact Factor
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ABSTRACT: Vascular smooth muscle (VSMC) and endothelial cells (EC) are particularly resistant to infection by type 5 adenovirus (Ad) vectors. To overcome this limitation and target Ad vectors to ubiquitously expressed alpha(V)beta(3/5) integrins, we have generated a linker protein consisting of the extracellular domain of the coxsackie adenovirus receptor (CAR) connected via avidin to a biotinylated cyclic (c) RGD peptide. After optimization of CAR to cRGD and to Ad coupling, infection of mouse heart endothelial cells (H5V) could be augmented significantly, as demonstrated by 600-fold increased transgene expression levels. In EOMAs, a hemangioendothelioma-derived cell line, the fraction of infected cells was enhanced 4- to 6-fold. Furthermore, the fraction of infected primary mouse VSMC was increased from virtually 0% to 25%. Finally, in human umbilical vein endothelial cells, the number of GFP positive cells was enhanced from 2% to 75%. In conclusion, CAR-cRGD is a versatile and highly efficient construct to target Ad vectors to both transformed and primary VSMC and EC.Biochemical and Biophysical Research Communications 01/2006; 338(2):847-54. DOI:10.1016/j.bbrc.2005.10.073 · 2.28 Impact Factor