Direct DNA transfer

1
Direct DNA transfer

• Introduce DNA into cells assay expression
  immediately or select for permanently
  transformed cells.
• Techniques
• Chemical induction
• Electroporation
• Particle bombardment (Biolistics)

2
Chemically-Induced Transformation

• Typically used on cells without walls
• There are multiple protocols
• Examples
• Put DNA inside artificial membrane vesicles
  called liposomes, which will fuse with the plasma
  membrane of recipient cells, delivering the
  cargo.
• Bind DNA with polycations that neutralize the
  charged, sugar-PO4 backbone, and condense the
  DNA. Some cell types will endocytose this
  complex.
• Combine (1) and (2)

3
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

4
Particle Bombardment

• Less limitations than electroporation
• Can use on cells with walls
• Can transform organelles!
• Method
• Precipitate DNA onto small tungsten or gold
  particles.
• Accelerate particles to high speeds and aim them
  at cells or tissues.
• Selective growth and regeneration of transgenic
  plants as described for Agro-mediated
  transformation.

5
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
6
An Air Rifle for a DNA Gun Circa 1990
A.Thompson, Bob ?, and D. Herrin
7
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
8
The Helium Gas Gun Circa 2000
9
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.
10
Transgenic Plants In Use on a Large Scale

• Herbicide-resistant plants
• Pest-resistant plants
• Vaccine plants (just starting to be used)

11
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

12
The function of EPSP synthase is to combine the
substrate shikimate-3-phosphate (S3P) with
phosphoenolpyruvate (PEP) to form
5-enolpyruvylshikimate-3-phosphate (EPSP).
13
Pest-resistant plants
Cry5

• Resistant to certain insects
• 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).
14
Insecticide Usage on Bt and non-Bt Crops for
1999-2001
15
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.
16
Concerns that have been raised about
cultivating/consuming GM crops (or GMOs)

• They may be toxic or allergenic.
• They may become established in the wild and
  outcompete other plants.
• They may negatively affect insects or other
  organisms that use crops.
• They may outcross to a nearby wild relative
  spreading the transgene into a wild population.

17
References on regulation and eco-risk 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