Making Transgenic Plants and Animals. Why? Study gene function and regulation Making new organismic tools for other fields of research Curing genetic diseases Improving agriculture and related raw materials New sources of bioengineered drugs (use plants instead of animals or bacteria).
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tumefaciens- causes crown galls on many dicots
rubi- causes small galls on a few dicots
rhizogenes- hairy root disease
More about Galls:
auxA auxB cyt ocs
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
These genes have typical eukaryotic expression signals!
Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485
Engineering plants with Agrobacterium:
Two problems had to be overcome:
(1) Ti plasmids large, difficult to manipulate
(2) couldn't regenerate plants from tumors
1. Move T-DNA onto a separate, small plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin resistance) gene in T-DNA.
4. Vir genes are retained on a separate plasmid.
5. Put foreign gene between T-DNA borders.
6. Co-transform Agrobacterium with both plasmids.
7. Infect plant with the transformed bacteria.
Making a transgenic plant by leaf disc transformation with Agrobacterium.
S.J. Clough, A.F. Bent (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743.
DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.
J. Sanford & T. Klein, 1988
A.Thompson, Bob ?, and D. Herrin
Repairing an organellar gene: ~ 1 x 107 cells of a mutant of Chlamydomonas that had a deletion in the atpB gene for photosynthesis was bombarded with the intact atpB gene. Then, the cells were transferred to minimal medium so that only photosynthetically competent cells could grow.
Control plate – cells were shot with tungsten particles without DNA
Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue)
One interesting use:
Making DNA Vaccines in whole animals.
The function of EPSP synthase is to combine the substrate shikimate-3-phosphate (S3P) with phosphoenolpyruvate (PEP) to form 5-enolpyruvylshikimate-3-phosphate (EPSP).
A Transmission Electron Micrograph of negatively stained spores from Bt2-56 containing a filament (a), and a sac-like structure containing a spore (b) and parasporal body (c).
C.J. Arntzen et al. (2005) Plant-derived Vaccines and Antibodies: Potential and Limitations. Vaccine 23, 1753-1756.