Adenovirus-mediated gene transfer
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 693:321-43. DOI: 10.1007/978-1-60761-974-1_20
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.
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ABSTRACT: The ion transport abnormalities in cystic fibrosis are becoming increasingly well defined, although how these lead to lung pathology is still speculation. Correction of these defects could theoretically be achieved either through pharmacological means or via gene therapy. Pharmacological approaches include increasing the amount of CFTR protein that reaches its correct localisation in epithelial cells. Secondly, approaches have been suggested which could increase the function of the protein already present at this correct localisation. Finally, it may be possible to identify alternative channels which could subserve the function of CFTR. Gene therapy is theoretically an attractive proposition as it should circumvent each of the identified abnormalities in cystic fibrosis. The principal difficulty at present relates to delivering sufficient copies of the normal CFTR gene into the appropriate cell population in vivo. A number of clinical trials have now been undertaken and steady and encouraging progress has been made in moving this approach from theory to practice.
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ABSTRACT: Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.
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ABSTRACT: Therapeutic angiogenesis achieved either through the use of discreet angiogenic proteins or by gene therapy is fast emerging as a highly attractive treatment modality for ischemic heart disease. Herein we examine a novel method of stimulating myocardial angiogenesis by hypoxic preconditioning at both capillary and arteriolar levels, and the potential role of NF kappa B in mediating such a response. We also investigate the functional relevance of such treatment by assessing whether the induced neovascularization can help preserve left ventricular contractile functional reserve in the setting of developing heart failure secondary to myocardial infarction. Male Sprague-Dawley rats were randomly divided into eight groups: normoxia + sham surgery (NS), normoxia + permanent left anterior descending coronary artery (LAD) occlusion (NMI), hypoxic preconditioning + sham surgery (HS), hypoxic preconditioning + permanent LAD occlusion (HMI), PDTC (NF kappa B inhibitor) + hypoxic preconditioning + LAD occlusion (PHMI), PDTC+normoxia + LAD occlusion (PNMI), PDTC + hypoxic preconditioning + sham surgery (PHS) and PDTC + normoxia + sham surgery (PNS). Rats in the preconditioned groups were subjected to systemic hypoxemic hypoxic exposure (10+/-0.4% O2) for 4 h followed by a 24-h period of normoxic reoxygenation prior to undergoing LAD occlusion. Rats in the normoxia groups were time matched with the preconditioned group and maintained under normoxic conditions for the 28-h period prior to LAD occlusion. The HMI group displayed significant increases in capillary as well as arteriolar density after 2, 4 and 7 days post-operation compared to the NMI. Prior PDTC administration prevented such increases in the PHMI group and effectively abolished the pro-angiogenic effect of hypoxic preconditioning (HP). One week after sham surgery or LAD occlusion, rats underwent a pharmacological stress test with dobutamine in progressively increasing doses which revealed significantly elevated values of dp/dt(max) at each dose point in the HMI group compared to the NMI or PHMI groups. Hypoxic preconditioning also decreases endothelial cell injury as determined by the extent of endothelial cell apoptosis using anti-VWF factor labelling and TUNEL assay. The results suggest that HP stimulates myocardial angiogenesis via redox-regulated transcription factor, NF kappa B-dependent pathway to an extent sufficient to exert significant preservation of contractile functional reserve in a rat model of myocardial infarction progressing to heart failure.
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