Molecular Methods of cell culture I
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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
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 cloning3. 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
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
DNA
Cells
Electroporator
http://www.jove.com/details.php?id=240
Transfecting Human Neural Stem Cells with the Amaxa Nucleofector
http://www.youtube.com/watch?v=ulA8xsVji80
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, 161-183; doi:10.3390/ph6020161
(-CH2-CH2-CONH-CH2-CH2-N-)
Tumor homing peptideiron oxide nanoparticles
(SPION) Stabilized by PEG
Pharmaceuticals 2013, 6, 161-183; doi:10.3390/ph6020161
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 transferliposome fuse directly with cell membrane and delivers DNA into cells
http://www.azonano.com/article.aspx?ArticleID=1233
http://www.youtube.com/watch?v=cPA2OQv8qA8
Lipofectamine transfection
3. Microinjection: direct injection of DNA in to nucleus
http://www.jove.com/details.php?id=1614
Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons
http://www.youtube.com/watch?v=M1-N9S84ydA&feature=related
Microinjection
Electrical
Toxicity
chemical
mechenical/electrical
Delivery efficiency
controllrd release polymers
Naked DNAmicroinjection
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 selectionDrug 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
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