Molecular%20Methods%20of%20cell%20culture%20I

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Molecular Methods of cell culture I
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DNA Delivery Pathway
Luo etal Nature biotechnology vol18 , 2000
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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
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Incorporate into plasmid with selectable marker
with restriction endonuclease
Extract DNA or RNA and prepare cDNA
Trasfection into recipient cells with
lipofection, calcium phosphate or electroporation
Grow up cells transfected cells in selective
medium, and assay for expression
Clone in bacteria in selective condition
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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
http//www.youtube.com/watch?vqx72xt0utm4
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DNA Transfection Methods Mechanical ?
Microinjection ? Pressure ? Particle bombardment
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• 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

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Electric Pulse
Membrane open
DNA enter
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Cuvette for Electroporation
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Cells
DNA
Electroporator
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Electroporation
http//www.youtube.com/watch?vulA8xsVji80
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• Chemical
• ? DEAE (diethylaminoethyl)?????? dextran
• ? Calcium phosphate
• ? Artificial lipid
• Proteins
• ? Polylysine( PLL). condense DNA
• ? Gal4 Invasin Poly-lysine

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• Dendrimers Dendron tree
  merospart,
• 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
doi10.3390/ph6020161
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A cancer cell-targeted dendrimer-siRNA-SPION
complex.
iron oxide nanoparticles (SPION)
Tumor homing peptide
Stabilized by PEG
Pharmaceuticals 2013, 6, 161-183
doi10.3390/ph6020161
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• Other polymers( including control release
  polymers)
• ? encapsulate naked DNA into PLGApoly(D,L-lactid
  e-co-glycolide)

??
???
Chemical structure of poly(D,L-lactide-co-glycolid
e) and its degradation products. m and n
refer to the relative amounts of lactide and
glycolide units respectively in a specific PLGA
copolymer.
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2. Liposomediated gene transfer ?liposome fuse
directly with cell membrane and delivers DNA into
cells
http//www.azonano.com/article.aspx?ArticleID1233
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Lipofectamine transfection
http//www.youtube.com/watch?vcPA2OQv8qA8
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3. Microinjection direct injection of DNA in to
nucleus
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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
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Transient transfection
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2. Stable Ttransfection ? Clone selected by
G418 ( geneticin) or hygromycin ? may be used to
obtain high protein expression by gene
amplification
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Stable Transfection
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Dominant selectable markers Used in transfection
experiments 1.Aminoglycoside phosphotransferase(A
PH) ? G418( inhibit protein synthesis.)
? APH inactivate G418 2.Dihydrofolate reductase
(DHFR)Mtx-resistant ? Methorexate( inhibit
DHFR) ? variant DHFR resist to Mtx
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Aminoglycoside posphotransferase (APH)
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2.Dihydrofolate reductase (DHFR) Mtx-resistant
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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