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

In der vorliegenden Arbeit wurde ein Assay zur Untersuchung von PEIs als nicht-virale Vektoren für siRNA entwickelt. Hierbei diente PEI 25kDa als Kontrolle. Des Weiteren wurde PEI 5kDa (Low-Molecular-Weight-PEI „LMW-PEI“) mit geringerer Verzweigung, PEI(25k)-g-PEG(550)35 (pegyliert) mit schwächerer...

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Bibliographic Details
Main Author: Merkel, Olivia Monika
Format: Work
Language:German
Published: Philipps-Universität Marburg 2006
Pharmazeutische Technologie und Biopharmazie
Subjects:
3T3
Online Access:PDF Full Text
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Table of Contents: 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.