Transcript Direct DNA transfer - University of Texas at Austin

Direct DNA transfer
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Introduce DNA into cells; assay expression
immediately or select for permanently
transformed cells.
Techniques:
1. Chemical induction
2. Electroporation
3. Particle bombardment (Biolistics)
Chemically-Induced Transformation
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Typically used on cells without walls
There are multiple protocols
Examples:
1. Put DNA inside artificial membrane vesicles
called liposomes, which will fuse with the
plasma membrane of recipient cells,
delivering the cargo.
2. Bind DNA with polycations that neutralize the
charged, sugar-PO4 backbone, and
condense the DNA. Some cell types will
endocytose this complex.
3. Combine (1) and (2)
Electroporation
• Use on cells without walls
(plant protoplasts or animal
cells).
• High-voltage pulses cause
pores to form transiently in
cell membrane; DNA pulled
in by electrophoresis and
diffusion.
• Drawback - its more
cumbersome to regenerate
plants from single
protoplasts than from the
tissue transformations with
Agrobacterium
Particle Bombardment
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Less limitations than electroporation
Can use on cells with walls
Can transform organelles!
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Method:
1. Precipitate DNA onto small tungsten or gold
particles.
2. Accelerate particles to high speeds and aim them at
cells or tissues.
3. Selective growth and regeneration of transgenic
plants as described for Agro-mediated
transformation.
Original 22-caliber biolistic gun
DNA is bound to the microprojectiles, which impact
the tissue or immobilized cells at high speeds.
J. Sanford & T. Klein, 1988
An Air Rifle for a DNA Gun –
Circa 1990
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
The Helium Gas Gun – Circa 2000
The Hand-Held Gas Gun
Purpose:
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.
Transgenic Plants In Use on a Large
Scale
• Herbicide-resistant plants
• Pest-resistant plants
• Vaccine plants (just starting to be used)
Herbicide-resistant plants
• Resistant to herbicide “Round-up” (glyphosate),
which inhibits EPSP synthase.
• GEngineered plants contain a bacterial EPSP
synthase, which is not inhibited by glyphosate.
• Advantages: better weed control, less tillage
• In use: soybeans (dicot), corn, rice, wheat
The function of EPSP synthase is to
combine the substrate shikimate-3phosphate (S3P) with
phosphoenolpyruvate (PEP) to form 5enolpyruvylshikimate-3-phosphate
(EPSP).
Pest-resistant plants
• Resistant to certain insects
Cry5
– Lepidopterans, Coleopterans
• Carry gene(s) for Bacillus
thuringiensis (Bt) toxin
• Toxin proteins produced as a
parasporal crystal
– crystalline material has several
proteins
– Cry and Cyt genes
– encoded on a plasmid
• Advantage: less insecticide
required, better yield
• corn, cotton, potatoes
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).
Insecticide Usage on Bt and non-Bt Crops for 1999-2001
Vaccine plants
• pioneered by Charlie Arntzen
• cheap vaccine-delivery system
• plant produces protein(s) or DNA from the human
pathogen, and immunity is induced via food
– potatoes, bananas
• being developed for a number of human and animal
diseases, including measles, cholera, foot and
mouth disease, and hepatitis B and C.
• Four plant vaccines were successful in phase I
clinical trials.
C.J. Arntzen et al. (2005) Plant-derived Vaccines and Antibodies: Potential and
Limitations. Vaccine 23, 1753-1756.
Concerns that have been raised
about cultivating/consuming GM
crops (or GMOs)
1. They may be toxic or allergenic.
2. They may become established in the wild
and outcompete other plants.
3. They may negatively affect insects or other
organisms that use crops.
4. They may outcross to a nearby wild relative
spreading the transgene into a wild
population.
References on regulation and ecorisk assessment vis-à-vis the
cultivation of GM crops
• Nap et al. (2003) Plant Journal 33, 1-18
– Focuses on current status and regulations
• Conner et al. (2003) Plant Journal 33, 19-46
– Focuses on ecological risk assessment
• GM Crops: A World View. Science, April
2008.