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WP5 progress and planning University of Helsinki

This project aims to investigate the interaction between various cell types and polymer materials, focusing on CPP-containing polyplexes and CIP-containing polymer membranes. The study includes transfection of differentiated ARPE19 cells with reporter genes to evaluate gene expression and secretion patterns. The research identifies parameters affecting transfection efficiency, such as cell density, polyplex preparation buffer, charge ratios, DNA amount, and incubation times. The optimized protocol will be standardized for all partners involved by the 18th month. Future activities include transfection of retinal cells on polymer membranes, evaluation of transfection efficiency using functionalized polymer membranes, and transfection with therapeutic genes.

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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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