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Transfection of eukaryotic cells

Transfection of eukaryotic cells. Bryon Anderson. Presentation Overview. What is Transfection? Transfection vs. Transformation Purpose of Transfection How it Works Experimental method/process Strengths and weaknesses of technique. Transfection. What is Transfection?

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Transfection of eukaryotic cells

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  1. Transfection of eukaryotic cells Bryon Anderson

  2. Presentation Overview • What is Transfection? • Transfection vs. Transformation • Purpose of Transfection • How it Works • Experimental method/process • Strengths and weaknesses of technique

  3. Transfection • What is Transfection? Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods. • Methods: (just a few examples…) MethodApplication CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection

  4. Transfection vs. Transformation • Transformation: genetically altering cells by transporting in foreign genetic material. • Transfection: the process of transporting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.

  5. Purpose/uses of Transfection • Study gene function • Study protein expression • Transfer DNA into embryonic stem cells

  6. How it works • Utilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.

  7. How it works – Chemical and lipid methods • Neutralize negative charges on DNA • Chemical method: Calcium phosphate; creates precipitates that settle on cells and are taken in. • Lipid or Polymer methods: interact with DNA, promotes binding to cells and uptake via endocytosis.

  8. How it works – Physical methods • Electroporation: use of high voltage to deliver nucleic acids; pores are formed on cell membrane. • Direct Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells. • Biolistic Particle delivery: Uses high-velocity for delivery of nucleic acids and penetration of cell wall.

  9. Experimental method/process(chemical methods) HBS = HEPES-Buffered Saline

  10. Advantages: Provides the ability to transfer in negatively charged molecules into cells with a negatively charged membrane. Liposome-mediated transport of DNA has high efficiency. Good for long-term studies requiring incorporation of genetic material into the chromosome. Disadvantages: Chemical Reagents: not useful for long-term studies. Transfection efficiency is dependent on cell health, DNA quality, DNA quantity, confluency (40-80%) Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method. Advantages/Disadvantages

  11. Another interesting use • “…dermal patches consisting of gene-transfected cultured skin, which secrete endogenous antimicrobial peptides such as B-defensins instead of exogenous antibiotics, can be a new DDS for the treatment of severe burns without decrease in cell viability.” Nobuko Hada, Hiroaki Todo, Fusao Komada, & Kenji Sugubayashi. (2007). Preparation and Evaluation of Gene-Transfected Cultured skin as a Novel Drug Delivery System for Severely Burned Skin. Pharmaceutical Research, Vol. 24, No. 8, p.1473-1479.

  12. References Pictures Used: (in order of appearance) http://www.nordicbiosite.dk/uploads/Transfection.gif Stratagene Transfection Reagents list (see below) Protocols and Applications Guide: www.promega.com http://www.fao.org/docrep/004/t0094e/t0094e03.htm http://electroporation.eu/ http://www3.bio-rad.com http://www.clontech.com/products/detail.asp?tabno=2&catalog_id=631312&page=all http://fredcobio.wordpress.com/2008/03/18/dna-vaccine-delivery-program-at-nci/ http://journals.cambridge.org/fulltext_content/ERM/ERM5_22/S1462399403006562sup002.htm

  13. References Information: • Transfection Reagents: http://www.stratagene.com/lit_items/Transfection_Brochure_BR61__Web.pdf • Protocols and Applications Guide: www.promega.com • http://www.iscid.org/encyclopedia/Transfection_Methods • Nobuko Hada, Hiroaki Todo, Fusao Komada, & Kenji Sugubayashi. (2007). Preparation and Evaluation of Gene-Transfected Cultured skin as a Novel Drug Delivery System for Severely Burned Skin. Pharmaceutical Research, Vol. 24, No. 8, p.1473-1479. • Menuel S, Fontanay S, Clarot I, Duval R.E, Diez L, Marsura A (2008). "Synthesis and Complexation Ability of a Novel Bis- (guanidinium)-tetrakis-(β-cyclodextrin) Dendrimeric Tetrapod as a Potential Gene Delivery (DNA and siRNA) System. Study of Cellular siRNA Transfection". Bioconjugate Chem.19 (12): 2357–2362. doi:10.1021/bc800193p.

  14. Questions?

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