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Molecular Methods of cell culture I

Molecular Methods of cell culture I. Low uptake. slow release of constructs with limited stability. Lack of nuclear targeting. Escape from endosome. endocytosis. Intracellular degradation. endosome. DNA Delivery Pathway. Luo etal Nature biotechnology vol18 , 2000.

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Molecular Methods of cell culture I

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  1. Molecular Methods of cell culture I

  2. Low uptake slow release of constructs with limited stability Lack of nuclear targeting Escape from endosome endocytosis Intracellular degradation endosome DNA Delivery Pathway Luo etal Nature biotechnology vol18 , 2000

  3. 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

  4. Incorporate into plasmid with selectable marker with restriction endonuclease Extract DNA 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 Clone in bacteria in selective condition

  5. The first methods used for DNA transfection 1. DEAE( Diethylamine ethyl)  positively charged  enter cells by endocytosis 乙基二乙胺 2. Calcium Phosphate  Divalent cations promote DNA entry in bacterial cells http://www.youtube.com/watch?v=qx72xt0utm4

  6. DNA Transfection Methods Mechanical  Microinjection  Pressure  Particle bombardment

  7. 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

  8. Electric Pulse Membrane open DNA enter

  9. Cuvette for Electroporation

  10. Cells DNA Electroporator

  11. 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

  12. Chemical  DEAE (diethylaminoethyl)二乙氨基乙基dextran Calcium phosphate Artificial lipid • Proteins  Polylysine( PLL)……. condense DNA  Gal4+ Invasin+ Poly-lysine

  13. DendrimersDendron : treemeros: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( PAMAMas carrier for siRNA delivery (-CH2-CH2-CONH-CH2-CH2-N-) Pharmaceuticals 2013, 6, 161-183; doi:10.3390/ph6020161

  14. A cancer cell-targeted dendrimer-siRNA-SPION complex. iron oxide nanoparticles (SPION) Tumor homing peptide Stabilized by PEG Pharmaceuticals 2013, 6, 161-183; doi:10.3390/ph6020161

  15. 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.

  16. 2. Liposomediated gene transfer liposome fuse directly with cell membraneand delivers DNA into cells http://www.azonano.com/article.aspx?ArticleID=1233

  17. Lipofectamine transfection http://www.youtube.com/watch?v=cPA2OQv8qA8

  18. 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

  19. Comparison of Transfection Methods Electrical high voltage electroporation chemical Toxicity mechenical/electrical Low voltage electroporation controllrd release polymers microinjection Naked DNA Delivery efficiency

  20. 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

  21. Transient transfection

  22. 2. Stable Ttransfection  Clone selected by G418 ( geneticin) or hygromycin  may be used to obtain high protein expression by gene amplification

  23. Stable Transfection Drug selection Clone selected

  24. Dominant selectable markers Used in transfection experiments 1.Aminoglycoside phosphotransferase(APH)  G418( inhibit protein synthesis.)  APH inactivate G418 2.Dihydrofolate reductase (DHFR):Mtx-resistant  Methorexate( inhibit DHFR)  variant DHFR resist to Mtx DHFR APH

  25. Aminoglycoside posphotransferase (APH)

  26. 2.Dihydrofolate reductase (DHFR) :Mtx-resistant

  27. 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

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