Transgenics: Direct DNA Transfer

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Transgenics Direct DNA Transfer

• Introduce naked DNA into cells (plant or animal)
• Can assay expression of the gene immediately, or
  select cells that are permanently transformed
  cells
• DNA introduction methods
• Chemical
• Microinjection
• Electroporation
• Particle bombardment (Biolistics)

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Chemically-induced transformation

• Usually use on cells without walls
• Multiple protocols
• put DNA inside artificial membranes (liposomes),
  they will fuse with plasma membrane
• Bind DNA with polycations to neutralize charge,
  some cells endocytose the complex
• Combine (2) and (1)

3
needle
Microinjection of DNA into the pronucleus of a
newly fertilized egg. Injection is usually into
the sperms pronucleus because its larger.
1-2 picoliter vol is injected. 5-40 of
animals will contain transgene.
From Primrose, Molec. Biotechnology
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Electroporation

• Use on cells without walls (plant protoplasts or
  animal cells )
• Used on monocots (maize, rice, etc.)
• High-voltage pulses cause pores to form
  transiently in cell membrane, DNA slips in
• Drawback - its more cumbersome to regenerate
  plants from single protoplasts than from the
  tissue transformations with Agrobacterium

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Particle Bombardment (Biolistics)

• Less limitations than electroporation
• Can use on cells with walls, or essentially any
  tissue
• Can transform organelles
• Method
• Precipitate DNA onto small tungsten or gold
  particles.
• Accelerate particles to high speeds to penetrate
  cells and tissues.
• Perform selective growth and regeneration of
  transgenic plants as described for Agro-mediated
  transformation.

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Original biolistic gun, a modified .22
DNA is bound to the microprojectiles, which are
accelerated by the macroprojectile and impact the
tissue or immobilized cells at high speeds.
J. Sanford and T. Klein, Cornell
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An Air Rifle for a DNA Gun Circa 1989
A.J. Thompson and D. Herrin
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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
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The Helium Gas Gun Circa 2000
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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.
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Transgenic Plants In Use or About to be on a
Large Scale

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

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Herbicide-resistant plants

• Resistant to herbicide Round-up (Glyphosate)
• Contain bacterial EPSP synthase
• Advantages better weed control, less tillage
• soybeans, corn, rice, wheat

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Pest-resistant plants

• Resistant to certain insects
• Plants carry gene(s) for Bacillus thuringiensis
  (Bt) toxin
• Advantage less insecticide required, better
  yield
• corn, cotton, potatoes

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Vaccine plants

• cheap vaccine-delivery system
• use plants producing pathogen protein to induce
  immunity
• potatoes, bananas

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Concerns that have been raised about cultivating
and consuming GM crops

• 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.

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References on release of GM crops into the
environment

• 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