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Presentation Gene Delivery
- 1. Instituto Superior Técnico 2009/2010 Mestrado em Bioengenharia e Nanossistemas Projecto Integrado I David Conceição nº64405
- 2. Gene delivery/therapy Gene delivery/therapy Magneto- Gene therapy is a technique for correcting defective fection genes responsible for disease development. HEK293 cell line A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target Cell cells. culture Plasmid DNA protect the transgene against degradation by transfection nucleases in intercellular matrices; Results & 3 major bring the transgene across the plasma Discussion criteria membrane and into the nucleus of target cells; no detrimental effects Conclusion Projecto Integrado I, July 2010
- 3. Gene delivery/therapy Gene delivery/therapy Viral vectors Magneto- fection Retroviruses; HEK293 cell line Adenoviruses; Adeno-associated Cell viruses (AAV); culture Herpes simplex viruses (HSV) Plasmid DNA transfection Results & Discussion Efficient gene transfer; Host’s reaction; Conclusion Unfavorable Pharmaceutical issue-large scale production Projecto Integrado I, July 2010
- 4. Gene delivery/therapy Gene delivery/therapy Non-Viral vectors Magneto- fection HEK293 Physical methods (carrier-free gene delivery) cell line Cell culture Chemical methods (synthetic vector-based gene delivery Plasmid DNA transfection Results & Discussion Conclusion Projecto Integrado I, July 2010
- 5. Gene delivery/therapy Gene delivery/therapy Non-Viral vectors Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion Conclusion Projecto Integrado I, July 2010
- 6. Magnetofection Gene delivery/therapy Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection A magnetic force aroused from a magnetic field applied among magnetic particles and a magnetofactor plate induces a bigger Results & Discussion attraction between cells and the nucleic acids, or transfection reagents or virus associated with magnetic particles, targeting the Conclusion gene delivery without changing much the standard protocol. Projecto Integrado I, July 2010
- 7. Magnetofection Gene delivery/therapy Magneto- fection HEK293 cell line Cell culture Usable with all types of nucleic Plasmid DNA acids (plasmid DNA, RNA, siRNA, transfection ODN) Results & Designed to be associated with Discussion viruses, or with another commercial transfection reagent Conclusion These magnetofection reagents are composed with magnetic nanoparticles associated with a proprietary polymer Projecto Integrado I, July 2010
- 8. HEK293 cell line Gene delivery/therapy Magneto- fection Human embryonic kidney cells HEK293 cell line Transformation and culturing of normal HEK cells with sheared Cell adenovirus 5 DNA culture Plasmid DNA Incorporation of approximately 4.5 transfection kilobases from the viral genome into human chromosome 19 of the HEK cells Results & Discussion The name HEK293 is thusly named Conclusion because it was Frank Graham's 293rd experiment Projecto Integrado I, July 2010
- 9. Cell culture Gene delivery/therapy Magneto- Laminar Flow Hood fection Medium bottles HEK293 T flasks cell line Well plates Cell Pipettes culture Etc, Plasmid DNA transfection Culture medium: Dulbecco's Modified Eagle Results & Medium (DMEM) high Discussion glucose (4.5 g/l), 10% fetal bovine serum (FBS), Conclusion 1% penicillin/streptomycin (100 μg/ml) (Invitrogen) Projecto Integrado I, July 2010
- 10. Cell culture Gene delivery/therapy Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion *- Trypan blue is the most common stain used to distinguish viable cells from nonviable cells; Conclusion - Neubauer hemacytometer Projecto Integrado I, July 2010
- 11. Plasmid DNA transfection Gene delivery/therapy Plasmid Magneto- fection Vector carrier of the transgene of interest: HEK293 cell line - pcDNA 3.1 (Invitrogen) Cell Identification and measurement of the culture fluorescence level by flow cytometry using Plasmid DNA FACSCalibur equipment (BD Biosciences) - transfection Fluorescence Activated Cell Sorting Results & Discussion Conclusion Projecto Integrado I, July 2010
- 12. Plasmid DNA transfection Gene delivery/therapy With lipofectamine Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection - Complexes were prepared using a DNA (µg) to Lipofectamine™ 2000 (Invitrogen) (µl) ratio of 1:2 Results & Discussion 49 µl of Opti-MEM and 1 µl of DNA solution 20 min of incubation 48 µl of Opti-MEM and 2 µl of Lipofectamine (with 5 min of Conclusion incubation) 18-48h of incubation in the CO2 incubator Projecto Integrado I, July 2010
- 13. Plasmid DNA transfection Gene delivery/therapy With PolyMAG Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection The proper amount of DNA (1 µg = 1 µl of DNA plasmid solution) was diluted in 200 µl of Opti-MEM® I Medium Results & Discussion This solution was then added to the eppendorf containig 0.5 µl of PolyMAG and mixed gently before a 15 minutes incubation Conclusion DNA-PolyMAG complexes were added to each well containing cells and Opti- MEM® I Medium, and the plate was put over the magnetofector plate 30 min of incubation in the CO2 incubator Projecto Integrado I, July 2010
- 14. Plasmid DNA transfection Gene delivery/therapy With CombiMAG Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection Lipoplexes were formed as it was described previously Results & Discussion Lipoplexes were added to an eppendorf containing 0.5 µl of CombiMAG 15 minutes of incubation Conclusion The plate was put onto the magnetofactor plate and was left in the incubator for 30 minutes Projecto Integrado I, July 2010
- 15. Plasmid DNA transfection Gene delivery/therapy FACS analysis Magneto- fection -Re-suspension of cells and HEK293 centrifugation; cell line - Cell counting; Cell culture Trypsin Plasmid DNA PBS transfection Results & Discussion Cells transferred to FACS tubes and subsequent analysis Conclusion Projecto Integrado I, July 2010
- 16. Results & Discussion Gene delivery/therapy Magneto- fection HEK293 cell line Cell culture Control group for toxicity effects Plasmid DNA transfection Histogram plots of the fluorescence signal carried by the analyzed cells Results & FACS analysis YFP-negative and positive cells: Discussion M2 and M1 Mean value of fluorescence Conclusion Projecto Integrado I, July 2010
- 17. Results & Discussion Gene delivery/therapy Magneto- fection With lipofectamine HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion %Gated cells (in M1): 61,84 Conclusion Mean value of fluorescence: 5865,26 Projecto Integrado I, July 2010
- 18. Results & Discussion Gene delivery/therapy Magneto- fection With polyMAG HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion %Gated cells (in M1): 22,47 Conclusion Mean value of fluorescence: 4245,55 Projecto Integrado I, July 2010
- 19. Results & Discussion Gene delivery/therapy Magneto- fection With CombiMAG HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion %Gated cells (in M1): 46,71 Conclusion Mean value of fluorescence: 5156,33 Projecto Integrado I, July 2010
- 20. Results & Discussion Gene delivery/therapy Magneto- fection Transfection efficiency, HEK293 Cell Viability, Cell cell line Recovery and Yield Cell culture Plasmid DNA transfection Results & Discussion Conclusion Projecto Integrado I, July 2010
- 21. Results & Discussion Gene delivery/therapy Magneto- Lipoplexes and CombiMAG complexes have the more intense fection level of fluorescence thereby having the higher level of protein expression, and also the bigger amount of YFP positive cells HEK293 cell line Scherer et al, proposed the use of magnetofection using its associated concepts such the magnetic force to improve the Cell DNA attraction to cells culture Plasmid DNA transfection Results & Discussion Conclusion Projecto Integrado I, July 2010
- 22. Results & Discussion Gene delivery/therapy Magnetofection does not necessarily improve the overall performance of a Magneto- given standard gene transfer method in vitro, but its major advantage consists fection on the possibility of remotely controlled vector targeting in vivo HEK293 cell line As Wang et al proposed, the preparation of Cell magnetic nanoparticles culture with other transfection reagents like cationic Plasmid DNA polymers or cationic transfection liposomes could be a great solution. Results & Discussion Conclusion Projecto Integrado I, July 2010
- 23. Conclusion Gene delivery/therapy Magneto- fection CombiMAG nanoparticles associated with lipoplexes gives interesting results (specially for a possible application in HEK293 cell line in vivo gene delivery with enhanced targeting). Cell culture Regarding cell viability, PolyMAG exhibited significant improval towards the other methods Plasmid DNA transfection This work gives a general readout for potential benefits and disadvantages but, for more coherent conclusions, Results & Discussion much more data must be collected Conclusion Projecto Integrado I, July 2010
- 24. Additional features Gene delivery/therapy Magneto- fection HEK293 cell line Cell culture Plasmid DNA transfection Results & Discussion Conclusion In vivo magnetofection Projecto Integrado I, July 2010