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