vrije universiteit brussels ipmb (2010/2011) introducing genes in eukaryotes by ngoe samuel bakia
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VRIJE UNIVERSITEIT BRUSSELS
IPMB (2010/2011)
INTRODUCING GENES IN EUKARYOTESINTRODUCING GENES IN EUKARYOTES
BYNGOE SAMUEL BAKIA
Major Strategies of TransformationMajor Strategies of Transformation
ChemicalChemicalTransfectionTransfection PhysicalPhysical
BactofectionBactofectionInfectionInfection TransductionTransduction
Overview of Transfortion MethodsOverview of Transfortion Methods
ApproachApproach MethodMethod
BiologicalBiological BacteriaBacteriaVirusesViruses
ChemicalChemical-Cationic molecules usedCationic molecules used-DNA taken up by DNA taken up by endocytosisendocytosis
DEAE-dextranDEAE-dextranCalcium phosphateCalcium phosphateCationic LipidsCationic LipidsDendrimersDendrimersPolyaminesPolyamines
ElectricalElectrical ELectroporationELectroporation
MechanicalMechanical MicroinjectionMicroinjectionParticle bombardmentParticle bombardmentPressurePressure
Three Major Barriers Three Major Barriers
lack of nuclear targeting. (A) DNA–complex formation. (B) Uptake. (C) Endocytosislack of nuclear targeting. (A) DNA–complex formation. (B) Uptake. (C) Endocytosis
(endosome). (D) Escape from endosome. (E) Degradation (edosome. (F) Intracellular release.(G) Degradation (endosome). (D) Escape from endosome. (E) Degradation (edosome. (F) Intracellular release.(G) Degradation (cytosol. (H) Nuclear targeting. (I) Nuclear entry and expression(cytosol. (H) Nuclear targeting. (I) Nuclear entry and expression
Cells Types for Transformation Cells Types for Transformation
PlantsPlants- ProtoplastProtoplast- Roots and Roots and
ShootsShoots- CallusCallus- ChloroplastChloroplast- Intact plantIntact plant
AnimalsAnimals- Embryonic Stem Embryonic Stem
CellsCells- Cell linesCell lines- Oocytes and Oocytes and
EggsEggs- Whole animal Whole animal
Viral Method: TransductionViral Method: Transduction
Bacterial delivery System: Bacterial delivery System: BactofectionBactofection
AgrobacteriumAgrobacterium-mediated plant transformatiom process-mediated plant transformatiom process
Chemical Method 1:DEAE-DextranChemical Method 1:DEAE-Dextran- DEAE-D associates with DEAE-D associates with
DNADNAcomplex binds to complex binds to
cell surfacecell surface
Add chloroquine to transfection mediumAdd chloroquine to transfection medium
Add plasmid DNA to DEAE-Dextran mediumAdd 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
ShockShock
Chemical Method 2: Calcium Chemical Method 2: Calcium PhosphatsePhosphatse
DNA mixed with calcium chloride, mixture added to DNA mixed with calcium chloride, mixture added to saline/phosphate buffer, precipitate dispersed onto culture cellssaline/phosphate buffer, precipitate dispersed onto culture cells
Chemical Method3: Cationic Chemical Method3: Cationic Lipids or liposomeLipids or liposome
Chemical Method 4: Use of DendrimerChemical 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 complexCondenses in star-shaped complex
Complex bind to cell membraneComplex bind to cell membrane
Complex taken up by endocytosisComplex taken up by endocytosis
Core space for molecular cargo Core space for molecular cargo
(DNA, RNA)(DNA, RNA)
Electrical Method: Electrical Method: ElectroporationElectroporation
Electric pulse perturb cell membraneElectric pulse perturb cell membrane
Nanopores created allowing entry of nucleic Nanopores created allowing entry of nucleic acidacid
Mechanical Method 1: MicroinjectionMechanical Method 1: Microinjection
Fine needle used to deliver nucleic acid directly Fine needle used to deliver nucleic acid directly inside the cellinside the cell
Mechanical Method 2: Biolistic Particle Mechanical Method 2: Biolistic Particle DeliveryDelivery
- Also called particle bombardment Also called particle bombardment - Cells bombarded with high velocity Cells bombarded with high velocity
microprojectilesmicroprojectiles
REFERENCESREFERENCES
-Dan Luo and W. Mark Saltzman (2000) Synthetic DNA delivery systems, -Dan Luo and W. Mark Saltzman (2000) Synthetic DNA delivery systems, Nature Nature BiotechnologyBiotechnology 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: 6460-64646464
-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, 13-16Springer science18, 13-16
--Sandy B. Primrose, Richard M. Twyman and Robert W OldSandy B. Primrose, Richard M. Twyman and Robert W Old , Principles of gene , Principles of gene manipulations, manipulations, 66thth 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, 66thth edition Wiley-Blackwell, 5, 85- edition Wiley-Blackwell, 5, 85-8686
-http://cellsighttech.com/technology/clinical-imaging-fhbg.html
--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
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