Master’s thesis deals with the design and testing of methods of increasing the transfection efficiency in mammalian cells. In this work a polyethylenimine (PEI) transfection method was used. The transfection efficiency was tested using MDA-MB-231 breast cancer cell line and pcDNA3.1-GFP-hMT-TOPO vector. We focused on optimization of PEI (1-6 µg) and plasmid DNA (1-3 µg) concentration, transfection time (0-48 h), number of cells per 9 cm2 (100,000-2 million) and medium composition. A complex containing linear penetration peptides (CPPs) KALA and GALA was assembled. Concentration of CPPs (7,5-45 µg) was optimized and a zeta potential and a size of the complexes were investigated. Due to the formation of large aggregates (over 1200 nm), modification with polyethyleneglycol (PEGylation) of PEI was performed. With the resulting complexes (~15 nm), transfection was performed. Transfection and apoptosis induction rates were compared between PEI and PEGylated PEI and as the best was chosen conjugate of PEGylated PEI (3 µg) with 45 µg of GALA and 3 µg pDNA with efficiency of 75%.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:427211 |
| Date | January 2018 |
| Creators | Štěpánková, Hana |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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