Samuel Ngoe - IPMB Gazette

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14 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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VRIJE UNIVERSITEIT BRUSSELS
IPMB (2010/2011)
INTRODUCING GENES IN EUKARYOTES
INTRODUCING GENES IN EUKARYOTES
BY
NGOE SAMUEL BAKIA
Major Strategies of Transformation
Major Strategies of Transformation
                                       
                                       
Chemical
Chemical
Transfection
Transfection


Physical
Physical


Bactofection
Bactofection
Infection
Infection


Transduction
Transduction
Overview of Transfortion Methods
Overview of Transfortion Methods
Approach
Approach
Method
Method
Biological
Biological
Bacteria
Bacteria
Viruses
Viruses
Chemical
Chemical
-
Cationic molecules used
Cationic molecules used
-
DNA taken up by
DNA taken up by
endocytosis
endocytosis
DEAE-dextran
DEAE-dextran
Calcium phosphate
Calcium phosphate
Cationic Lipids
Cationic Lipids
Dendrimers
Dendrimers
Polyamines
Polyamines
Electrical
Electrical
ELectroporation
ELectroporation
Mechanical
Mechanical
Microinjection
Microinjection
Particle bombardment
Particle bombardment
Pressure
Pressure
Three Major Barriers
Three Major Barriers
lack of nuclear targeting. (A) DNA–complex formation. (B) Uptake. (C) Endocytosis
lack of nuclear targeting. (A) DNA–complex formation. (B) Uptake. (C) Endocytosis
(endosome). (D) Escape from endosome. (E) Degradation (edosome. (F) Intracellular release.(G) Degradation (cytosol. (H) Nuclear 
(endosome). (D) Escape from endosome. (E) Degradation (edosome. (F) Intracellular release.(G) Degradation (cytosol. (H) Nuclear 
targeting. (I) Nuclear entry and expression
targeting. (I) Nuclear entry and expression
Cells Types for Transformation
Cells Types for Transformation
Plants
Plants
-
Protoplast
Protoplast
-
Roots and
Roots and
Shoots
Shoots
-
Callus
Callus
-
Chloroplast
Chloroplast
-
Intact plant
Intact plant
Animals
Animals
-
Embryonic Stem
Embryonic Stem
Cells
Cells
-
Cell lines
Cell lines
-
Oocytes and
Oocytes and
Eggs
Eggs
-
Whole animal
Whole animal
Viral Method: Transduction
Viral Method: Transduction
Bacterial delivery System:
Bacterial delivery System:
Bactofection
Bactofection
Agrobacterium
Agrobacterium
-mediated plant transformatiom process
-mediated plant transformatiom process
Chemical Method 1:DEAE-Dextran
Chemical Method 1:DEAE-Dextran
-
DEAE-D associates with
DEAE-D associates with
DNA
DNA


complex binds to
complex binds to
cell surface
cell surface
Add chloroquine to transfection medium
Add chloroquine to transfection medium
Add plasmid DNA to DEAE-Dextran medium
Add plasmid DNA to DEAE-Dextran medium
Incubate cells with mixture…efficient transfection
Incubate cells with mixture…efficient transfection
results in 25-75% death
results in 25-75% death




Shock
Shock
Chemical Method 2: Calcium
Chemical Method 2: Calcium
Phosphatse
Phosphatse
DNA mixed with calcium chloride, mixture added to
DNA mixed with calcium chloride, mixture added to
saline/phosphate buffer, precipitate dispersed onto culture cells
saline/phosphate buffer, precipitate dispersed onto culture cells














Chemical Method3: Cationic 
Chemical Method3: Cationic 
Lipids or liposome
Lipids or liposome
Chemical Method 4: Use of Dendrimer
Chemical Method 4: Use of Dendrimer
positive external amines bind DNA
positive external amines bind DNA
surface amino group
surface amino group
Condenses in star-shaped complex
Condenses in star-shaped complex
Complex bind to cell membrane
Complex bind to cell membrane
Complex taken up by endocytosis
Complex taken up by endocytosis


Core space for molecular cargo
Core space for molecular cargo


(DNA, RNA)
(DNA, RNA)
Electrical Method: 
Electrical Method: 
Electroporation
Electroporation
Electric pulse perturb cell membrane
Electric pulse perturb cell membrane
Nanopores created allowing entry of nucleic acid
Nanopores created allowing entry of nucleic acid
Mechanical Method 1: Microinjection
Mechanical Method 1: Microinjection


Fine needle used to deliver nucleic acid directly
Fine needle used to deliver nucleic acid directly
inside the cell
inside the cell
Mechanical Method 2: Biolistic Particle
Mechanical Method 2: Biolistic Particle
Delivery
Delivery
-
Also called particle bombardment
Also called particle bombardment
-
Cells bombarded with high velocity
Cells bombarded with high velocity
microprojectiles
microprojectiles
REFERENCES
REFERENCES
-Dan Luo and W. Mark Saltzman (2000) Synthetic DNA delivery systems,
-Dan Luo and W. Mark Saltzman (2000) Synthetic DNA delivery systems,
Nature
Nature
Biotechnology
Biotechnology
18, 33-37 (December 2000)
18, 33-37 (December 2000)
-Danos O, Mulligan RC (1988): Safe and efficient generation of recombinant retroviruses
-Danos O, Mulligan RC (1988): Safe and efficient generation of recombinant retroviruses
with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 85: 6460-
with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 85: 6460-
6464
6464
-Gan, Carol. "Gene Gun Accelerates DNA-Coated Particles To Transform Intact Cells".
-Gan, Carol. "Gene Gun Accelerates DNA-Coated Particles To Transform Intact Cells".


The Scientist;
The Scientist;
Sep. 18, 1989, 3[18]:25
Sep. 18, 1989, 3[18]:25
.
.
-
-
Rob Desalle and Elisabeth Brown (2006)
Rob Desalle and Elisabeth Brown (2006)
. Genetic engineering, Principles and methods,
. Genetic engineering, Principles and methods,
Springer science18, 13-16
Springer science18, 13-16
-
-
Sandy B. Primrose, Richard M. Twyman and Robert W Old
Sandy B. Primrose, Richard M. Twyman and Robert W Old
, Principles of gene
, Principles of gene
manipulations,
manipulations,
6
6
th
th
edition Blackwell Publishing, chp 10-13
edition Blackwell Publishing, chp 10-13
-
-
T.A. Brown (2010)
T.A. Brown (2010)
Gene Cloning and DNA Analysis,
Gene Cloning and DNA Analysis,
6
6
th
th
edition Wiley-Blackwell, 5, 85-
edition Wiley-Blackwell, 5, 85-
86
86
-
http://cellsighttech.com/technology/clinical-imaging-fhbg.html
-
-
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF
-http://www.promega.com/paguide/chap12.htm
-http://www.promega.com/paguide/chap12.htm
-
http://wiki.biomine.skelleftea.se/wiki/index.php/Electroporation