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Komplexierung von siRNA zum spezifischen Knock Down von Luciferase in SKOV-3/Luc und 3T3 Zellen

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2007-01-24

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Philipps-Universität Marburg
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Abstract

In this thesis, the establishment of an assay for the examination of poly(ethyleneimines) as non-viral vectors for siRNA is described. Here, branched PEI 25kDa served as a control and PEI 5kDa Low-Molecular-Weight-PEI „LMW-PEI“), less branched, PEI(25k)-g-PEG(550)35 (pegylated), which shows weaker complexation, and ePEI (ethoxylated) with a smaller pKa and stronger protonation in the endosome were tested as well. Conditions of transfection and N/P-ratios of polyplexes were optimized and polyplexes were charakterized physico-chemically by analysis of complexation in agarose gel electrophoresis and determination of hydrodynamic diameters with DLS. Knock down efficiency after transfection of a constitutively luciferase-expressing cell line SKOV-3/Luc and after self-imposed transfection with pDNA, encoding luciferase, was compared and quantified by chemoluminescence assay. Specificity was proven by detection of unspecific silencing effects with a scrambeled sequence control siRNA. Complexes were localized by fluorescence labeling and confocal laser scanning microscopy.

Keywords

Nicht-virale Vektoren, Co-Transfektion, Polyethylenimin, Gentherapie, siRNA, RNAi, Nucleic acids, DNA and RNA bases, SKOV-3, Luciferase, Transfektion, PEI-g-PEG, PEG-PEI, siGL3, siRNA, Transfection, Gene therapy, 3T3, Luciferase, Poly(ethylene imine)

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https://open.uni-marburg.de/handle/10.17192/ed.2006.0001

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