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WP5 progress and planning University of Helsinki

WP5 progress and planning University of Helsinki. Astrid Subrizi, 18 December 2007. WP 5: Characterization of polyplex-cell and polymer membrane-cell interactions.

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WP5 progress and planning University of Helsinki

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  1. WP5 progress and planningUniversity of Helsinki Astrid Subrizi, 18 December 2007

  2. WP 5: Characterization of polyplex-cell and polymer membrane-cell interactions. • Objective: to study the interaction of different cell types, including RPE cells, vascular endothelial cells and smooth muscle cells, with the polymer materials. • Both the interaction between CPP-containing polyplexes and cells and the interaction between CIP-containing polymer membranes and cells will be investigated.

  3. Transfection of differentiated ARPE19 cells with pCMV-SEAP2 and pEpi-SEAP • A secreted reporter gene (SEAP) was used in order to evaluate duration and direction of the secretion of the expressed gene product. • EBNA is responsible for extrachromosomal maintenance and it ensures replication of plasmid once per cell cycle during S phase and segregation into the daughter cells. From Mannermaa E, Curr Eye Res. 30:345–353, 2005 For each carrier tested, gene expression lasted for more than 40 respectively 60 days.

  4. EBNA plasmid pCMV-SEAP2 EBNA plasmid pCMV-SEAP2 pCMV-SEAP2 EBNA plasmid

  5. Optimized transfection protocol Parameters affecting transfection efficiency were identified and clarified: • Cell density – 4’000, 8’000 and 20’000 cells/well. • Polyplex preparation buffer – mqH2O and Mes-Hepes buffered saline. • Charge ratios (polymer/DNA) – 4/1, 2/1 and 1/1. • Amount of DNA – 300 and 600 ng per well (96 wp). • Incubation time of polyplexes with cells – 30 min., 1, 2, 5, 12 and 24 hours. • Incubation time after transfection – 24, 48 and 72 hours. The optimized protocol will be the standard operating procedure for every partner.

  6. Achievements by month 18th • Transfections of differentiated ARPE19 monolayers with 2 carriers (polyplexes and lipoplexes) and 2 plasmids, including EBNA plasmid pEpi-SEAP. • Development of an optimized transfection protocol, to be used in future experiments by all the partners of the consortium.

  7. Planned activities (months 19-36) • Transfection of retinal cells seeded on polymer membranes (strategy A). • Evaluation of transfection efficiency of polymer membranes functionalized with polyplexes, using retinal cells (strategy B). • Transfection of retinal cells with therapeutic genes.

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