1. VRIJE UNIVERSITEIT BRUSSELS IPMB (2010/2011) INTRODUCING GENES IN EUKARYOTES BY NGOE SAMUEL BAKIA

2. Major Strategies of Transformation Chemical ChemicalTransfection Physical Physical Bactofection BactofectionInfection Transduction Transduction

3. Overview of Transfortion Methods ApproachMethod Biological BacteriaViruses Chemical - Cationic molecules used - DNA taken up by endocytosis DEAE-dextran Calcium phosphate Cationic Lipids DendrimersPolyamines Electrical ELectroporation Mechanical Microinjection Particle bombardment Pressure

4. Three Major Barriers 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 targeting. (I) Nuclear entry and expression

5. Cells Types for Transformation Plants - Protoplast - Roots and Shoots - Callus - Chloroplast - Intact plant Animals - Embryonic Stem Cells - Cell lines - Oocytes and Eggs - Whole animal

6. Viral Method: Transduction

7. Bacterial delivery System: Bactofection Agrobacterium-mediated plant transformatiom process

8. Chemical Method 1:DEAE-Dextran - DEAE-D associates with DNA  complex binds to cell surface Add chloroquine to transfection medium Add plasmid DNA to DEAE-Dextran medium Incubate cells with mixture…efficient transfection results in 25-75% death Shock Shock

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

10. Chemical Method3: Cationic Lipids or liposome

11. Chemical Method 4: Use of Dendrimer positive external amines bind DNA surface amino group Condenses in star-shaped complex Complex bind to cell membrane Complex taken up by endocytosis Core space for molecular cargo Core space for molecular cargo (DNA, RNA) (DNA, RNA)

12. Electrical Method: Electroporation Electric pulse perturb cell membrane Nanopores created allowing entry of nucleic acid

13. Mechanical Method 1: Microinjection Fine needle used to deliver nucleic acid directly inside the cell Fine needle used to deliver nucleic acid directly inside the cell

14. Mechanical Method 2: Biolistic Particle Delivery - Also called particle bombardment - Cells bombarded with high velocity microprojectiles

15. REFERENCES -Dan Luo and W. Mark Saltzman (2000) Synthetic DNA delivery systems, Nature Biotechnology 18, 33-37 (December 2000) -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- 6464 -Gan, Carol. "Gene Gun Accelerates DNA-Coated Particles To Transform Intact Cells". The Scientist; Sep. 18, 1989, 3[18]:25. -Rob Desalle and Elisabeth Brown (2006). Genetic engineering, Principles and methods, Springer science18, 13-16 - Sandy B. Primrose, Richard M. Twyman and Robert W Old, Principles of gene manipulations, 6 th edition Blackwell Publishing, chp 10-13 -T.A. Brown (2010) Gene Cloning and DNA Analysis, 6 th edition Wiley-Blackwell, 5, 85- 86 -http://cellsighttech.com/technology/clinical-imaging-fhbg.htmlhttp://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://wiki.biomine.skelleftea.se/wiki/index.php/Electroporationhttp://wiki.biomine.skelleftea.se/wiki/index.php/Electroporation