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