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 endosome Intracellular degradation 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 or RNA and prepare cDNA with restriction endonuclease Incorporate into plasmid with selectable marker Clone in bacteria in selective condition Trasfection into recipient cells with lipofection, calcium phosphate or electroporation Grow up cells transfected cells in selective medium, and assay for expression

The first methods used for DNA transfection 1. DEAE( Diethylamine ethyl)  positively charged  enter cells by endocytosis 2. Calcium Phosphate  Divalent cations promote DNA entry in bacterial cells 乙基二乙胺

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

DNA Cells Electroporator

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector Electroporation

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 Pharmaceuticals 2013, 6, ; doi: /ph (-CH2-CH2-CONH-CH2-CH2-N-)

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

 Other polymers( including control release polymers)  encapsulate naked DNA into PLGA …poly(D,L-lactide-co-glycolide) 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. 乙醇酸 乳酸

2. Liposomediated gene transfer liposome fuse directly with cell membrane and delivers DNA into cells

Lipofectamine transfection

3. Microinjection: direct injection of DNA in to nucleus Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons Microinjection

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

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 expression by gene amplification

Stable Transfection Clone selected Drug selection

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 APH 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