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Molecular Methods of cell culture I DNA Delivery Pathway Low uptake slow release of constructs with limited stability Lack of nuclear targeting Escape from endosome endocytosis Intracellular degradation endosome Luo etal Nature biotechnology vol18 , 2000 Three essential tools form the basis for studying the function of mammalian genes: 1. Isolation of a mammalian gene 2. Cloning and manupilate of mammalian genes by DNA cloning 3. The technique should be able to return the altered gene to cells to determine the function Extract DNA with restriction endonuclease or RNA and prepare cDNA Grow up cells transfected cells in selective medium, and assay for expression Trasfection into recipient cells with lipofection, calcium phosphate or electroporation Incorporate into plasmid with selectable marker Clone in bacteria in selective condition The first methods used for DNA transfection 1. DEAE( Diethylamine ethyl) 乙基二乙胺 positively charged enter cells by endocytosis 2. Calcium Phosphate Divalent cations promote DNA entry in bacterial cells http://www.youtube.com/watch?v=qx72xt0utm4 DNA Transfection Methods Mechanical Microinjection Pressure Particle bombardment Electrical Electroporation( high voltage) Electroporation( low voltage) a brief change of electric pulse discharges across the electrode, transiently open holes in cells Applications for electroporation DNA introduction Drug loading Tumor tissue drug delivery Localized gene therappy low energy cell killing Loading dyes and tracers into cells Release of intracellular compound Transdermal drug delivery Electric Pulse Membrane open DNA enter Cuvette for Electroporation Cells DNA Electroporator Electroporation http://www.youtube.com/watch?v=ulA8xsVji80 Transfecting Human Neural Stem Cells with the Amaxa Nucleofector http://www.jove.com/details.php?id=240 Chemical DEAE (diethylaminoethyl)二乙氨基乙基 dextran Calcium phosphate Artificial lipid Proteins Polylysine( PLL)……. condense DNA Gal4+ Invasin+ Poly-lysine Dendrimers Dendron : tree meros:part, a structure that consists of a central core molecule that acts as a root, from which a number of highly branched, tree-like arms originates in a symmetrical manner polyamidoamine( PAMAM as carrier for siRNA delivery (-CH2-CH2-CONH-CH2-CH2-N-) Pharmaceuticals 2013, 6, 161-183; doi:10.3390/ph6020161 A cancer cell-targeted dendrimer-siRNA-SPION complex. iron oxide nanoparticles (SPION) Stabilized by PEG Tumor homing peptide Pharmaceuticals 2013, 6, 161-183; doi:10.3390/ph6020161 Other polymers( including control release polymers) encapsulate naked DNA into PLGA…poly(D,L-lactide-co-glycolide) 乳酸 乙醇酸 Chemical structure of poly(D,L-lactide-co-glycolide) and its degradation products. ‘m’ and ‘n’ refer to the relative amounts of lactide and glycolide units respectively in a specific PLGA copolymer. 2. Liposomediated gene transfer liposome fuse directly with cell membrane and delivers DNA into cells http://www.azonano.com/article.aspx?ArticleID=1233 Lipofectamine transfection http://www.youtube.com/watch?v=cPA2OQv8qA8 3. Microinjection: direct injection of DNA in to nucleus Microinjection http://www.youtube.com/watch?v=M1-N9S84ydA&feature=related Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons http://www.jove.com/details.php?id=1614 Comparison of Transfection Methods Electrical high voltage electroporation chemical Toxicity mechenical/electrical Low voltage electroporation controllrd release polymers microinjection Naked DNA Delivery efficiency Exogenous DNA is Transiently or Stably Expressed 1. Transient Transfection DNA expressed immediately after transfection Assay by reporter i.e. C.A.T. :chloramphenical acetyl transferase RNA transcription i.e. northern blotting Transient transfection 2. Stable Ttransfection Clone selected by G418 ( geneticin) or hygromycin may be used to obtain high protein gene amplification expression by Stable Transfection Drug selection Clone selected Dominant selectable markers Used in transfection experiments 1.Aminoglycoside phosphotransferase(APH) G418( inhibit protein synthesis.) APH APH inactivate G418 2.Dihydrofolate reductase (DHFR):Mtx-resistant Methorexate( inhibit DHFR) variant DHFR resist to Mtx DHFR Aminoglycoside posphotransferase (APH) 2.Dihydrofolate reductase (DHFR) :Mtx-resistant 3.Hygromycin-B-Phoshotransferase (HPH) Hygromycin-B( inhibit protein synthesis) HPH inactivate hygromycin B 4.Thymidine kinase(TK) Aminopeterine( inhibits de novo purine and thymidylate) TK synthesize thymidylate HPH