Transfection of eukaryotic cells Bryon Anderson. Presentation Overview What is Transfection?...
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Transcript of Transfection of eukaryotic cells Bryon Anderson. Presentation Overview What is Transfection?...
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Transfection of eukaryotic cells
Bryon Anderson
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Presentation Overview
What is Transfection?Transfection vs. TransformationPurpose of TransfectionHow it WorksExperimental method/processStrengths and weaknesses of technique
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TransfectionWhat 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…) Method Application
CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection
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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.
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Purpose/uses of Transfection
Study gene functionStudy protein expressionTransfer DNA into embryonic stem cells
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How it works
Utilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.
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How it works – Chemical and lipid methods
Neutralize negative charges on DNAChemical 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.
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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.
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Experimental method/process
(chemical methods)
HBS = HEPES-Buffered Saline
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Advantages/Disadvantages
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.
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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.
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ReferencesPictures Used: (in order of appearance)
http://www.nordicbiosite.dk/uploads/Transfection.gifStratagene Transfection Reagents list (see below)Protocols and Applications Guide: www.promega.comhttp://www.fao.org/docrep/004/t0094e/t0094e03.htmhttp://electroporation.eu/http://www3.bio-rad.comhttp://www.clontech.com/products/detail.asp?
tabno=2&catalog_id=631312&page=allhttp://fredcobio.wordpress.com/2008/03/18/dna-vaccine-delivery-
program-at-nci/http://journals.cambridge.org/fulltext_content/ERM/ERM5_22/
S1462399403006562sup002.htm
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References Information:
Transfection Reagents: http://www.stratagene.com/lit_items/Transfection_Brochure_BR61__Web.pdfProtocols and Applications Guide: www.promega.comhttp://www.iscid.org/encyclopedia/Transfection_MethodsNobuko 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.
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Questions?