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Gene delivery techniques

Gene delivery techniques. Genetic Engineering of Plants. Must get DNA: into the cells integrated into the genome Expressed For (1) and (2), two main approaches for plants: Direct gene transfer Agrobacterium - mediated gene transfer

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Gene delivery techniques

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  1. Gene delivery techniques

  2. Genetic Engineering of Plants • Must get DNA: • into the cells • integrated into the genome • Expressed • For (1) and (2), two main approaches for plants: • Direct gene transfer • Agrobacterium - mediated gene transfer • For (3), use promoter that will direct expression when and where wanted.

  3. Commonly used gene delivery techniques • Microinjection • Electroporation • Microprojectiles • Agrobacterium tumifaciensmediated transfer

  4. Microinjection • Mechanical method of gene insertion • Very fine micropipette used to inject DNA molecule directly into the nucleus • Most commonly used for animal embryos • Not very successful in case of plants

  5. Microinjection Source:www.translatingscience.pbwork.com

  6. Electroporation • Electric current is provided to plant cells • Tiny holes are created • DNA molecules present in the liquid medium • Diffusion into the cells • Cells allowed to recover • Regeneration into whole plant

  7. Microprojectile/Gene Gun • Most commonly used for monocots • Tungsten or gold particles coated with DNA • Shot into the cells • http://passel.unl.edu/pages/animation.php?a=GeneGun.swf

  8. Agrobacterium tumefaciens • Mainly for engineering dicot plants; monocots generally • Some dicots more resistant than others (a genetic basis for this) • Complex bacterium – genome has been sequenced; 4 chromosomes; ~ 5500 genes • soil bacteria, gram-negative, related to Rhizobia species: tumefaciens- causes crown galls on many dicots rubi- causes small galls on a few dicots rhizogenes- hairy root disease radiobacter- avirulent

  9. A. tumefaciens Ti (Tumor inducing) Plasmid A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Most commonly found as small circular, double-stranded DNA molecules in bacteria

  10. Structure of Ti Plasmid

  11. Ti Plasmid containing T-DNA region

  12. T-DNA auxA auxB cyt ocs LB RB LB, RB – left and right borders (direct repeat) auxA + auxB– enzymes that produce auxin cyt– enzyme that produces cytokinin Ocs – octopine synthase, produces octopine

  13. Agrobacteriummechanism of plant infection • The transferred genes form the T-DNA (transferred DNA) region of the Ti plasmid (tumour inducing). • T-DNA is less than 10% of the whole plasmid, encodes only 3 or so genes and enters a plant that has at least 25000 genes

  14. Effect of T-DNA genes on plant cells • Changes the plant’s metabolism to produce food materials (opines) that only the bacterium can use • Produces the plant hormones auxin and cytokinin that remove the controls that normally limit cell division and cell expansion. • Result - cells showing altered metabolism multiply uncontrollably

  15. RESULT Even if the T-DNA originally change only one cell in the plant, because that cell divides uncontrollably and passes on those bacterial genes to all the new cells, there are soon millions of cells feeding the bacterium at the expense of the plant.

  16. Infection of a plant with A. tumefaciens and formation of a crown gall tumor.

  17. EM of A. tumefaciens attached to plant cell

  18. Vir (virulent) genes • On the Ti plasmid • Transfer the T-DNA to plant cell • Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes • virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

  19. Vir gene functions (cont.) • virA- transports AS into bacterium, activates virG post-translationally (by phosphoryl.) • virG- promotes transcription of other vir genes • virD2 - endonuclease/integrase that cuts T-DNA at the borders but only on one strand; attaches to the 5' end of the SS • virE2- binds SS of T-DNA & can form channels in artificial membranes • virE1 - chaperone for virE2 • virD2 & virE2 get T-DNA to the nucleus of plant cell • virB- operon of 11 proteins, gets T-DNA through bacterial membranes

  20. A. tumifaciensmediated gene transfer Removal & Replacement of T-DNA region

  21. Engineered A. TumefaciensTi Plasmid vector

  22. Common Transformation Protocols • Leaf-disc transformation - after selection and regeneration with tissue culture, get plants with the introduced gene in every cell • Floral Dip – does not require tissue culture. Reproductive tissue is transformed and the resulting seeds are screened for drug-resistant growth (Clough and Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743)

  23. T-DNA transfer mechanism • ANIMATION http://highered.mheducation.com/sites/dl/free/0072835125/126997/animation39.html

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