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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