Making Transgenic Plants and Animals

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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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Genetic Engineering of Plants

• Must get DNA
• into the cells
• integrated into the genome (unless using
  transient expression assays)
• expressed (everywhere or controlled)
• For (1) and (2), two main approaches for plants
• Agrobacterium - mediated gene transfer
• Direct gene transfer
• For (3), use promoter that will direct expression
  when and where wanted may also require other
  modifications such as removing or replacing
  introns.

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Agrobacterium - mediated Gene Transfer

• Most common method of engineering dicots, but
  also used for monocots
• Pioneered by J. Schell (Max-Planck Inst.,
  Cologne)
• Agrobacteria
• 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

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Crown galls caused by A. tumefaciens on
nightshade.
More about Galls http//waynesword.palomar.edu/p
ljuly99.htm http//kaweahoaks.com/html/galls_ofthe
_voaks.html
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Agrobacterium tumefaciens

• the species of choice for engineering dicot
  plants monocots are generally resistant (but
  you can get around this)
• some dicots more resistant than others (a
  genetic basis for this)
• complex bacterium genome has been sequenced 4
  chromosomes 5500 genes

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Agrobacterium tumefaciens
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Infection and tumorigenesis

• Infection occurs at wound sites
• Involves recognition and chemotaxis of the
  bacterium toward wounded cells
• galls are real tumors, can be removed and will
  grow indefinitely without hormones
• genetic information must be transferred to plant
  cells

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Tumor characteristics

• Synthesize a unique amino acid, called opine
• octopine and nopaline - derived from arginine
• agropine - derived from glutamate
• Opine depends on the strain of A. tumefaciens
• Opines are catabolized by the bacteria, which
  can use only the specific opine that it causes
  the plant to produce.
• Has obvious advantages for the bacteria, what
  about the plant?

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Elucidation of the TIP (tumor-inducing principle)

• It was recognized early that virulent strains
  could be cured of virulence, and that cured
  strains could regain virulence when exposed to
  virulent strains suggested an extra- chromosomal
  element.
• Large plasmids were found in A. tumefaciens and
  their presence correlated with virulence called
  tumor-inducing or Ti plasmids.

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Ti Plasmid

• Large (?200-kb)
• Conjugative
• 10 of plasmid transferred to plant cell after
  infection
• Transferred DNA (called T-DNA) integrates
  semi-randomly into nuclear DNA
• Ti plasmid also encodes
• enzymes involved in opine metabolism
• proteins involved in mobilizing T-DNA (Vir genes)

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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
These genes have typical eukaryotic expression
signals!
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• auxA auxB
• Tryptophan? indoleacetamide ? indoleacetic acid
  (auxin)
• 
  cyt
• AMP isopentenylpyrophosphate ? isopentyl-AMP
  (a cytokinin)
• Increased levels of these hormones stimulate
  cell division.
• Explains uncontrolled growth of tumor.

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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.

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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 also have NLSs, gets T-DNA to the
  nucleus of plant cell
• virB - operon of 11 proteins, gets T-DNA
  through bacterial membranes

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From Covey Grierson
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• Type IV Secretion Sys.
• many pathogens, also used in conjugation
• promiscuous
• forms T-Pilus
• B7-B10 span OM IM
• B7-B9 in OM interacts w/B8 B10 of IM to form
  channel
• 3 ATPases
• D4 promotes specific transport
• B2 can form filaments

Gauthier, A. et al. (2003) J. Biol. Chem.
27825273-25276
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VirE2 may get DNA-protein complex across host PM
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA,
98485
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• Monocots don't produce AS in response to
  wounding.
• Important Put any DNA between the LB and RB of
  T-DNA it will be transferred to plant cell!

Engineering plants with Agrobacterium Two
problems had to be overcome (1) Ti plasmids
large, difficult to manipulate (2) couldn't
regenerate plants from tumors
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Binary vector system

• Strategy
• 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.

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Binary vector system
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2 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, 735743)

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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, 735743.
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Direct DNA transfer

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

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Chemically-Induced Transformation

• Usually use on cells without walls
• Multiple protocols (examples)
• 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 (1) and (2)

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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 or diffusion (?)
• Drawback - its more cumbersome to regenerate
  plants from single protoplasts than from the
  tissue transformations with Agrobacterium

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Particle Bombardment

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

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Original biolistic gun. A modified 22 caliber.
DNA is bound to the microprojectiles, which
impact the tissue or immobilized cells at high
speeds.
J. Sanford T. Klein, 1988
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An Air Rifle for a DNA Gun Circa 1990
A.Thompson, Bob ?, 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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The function of EPSP synthase is to combine the
substrate shikimate-3-phosphate (S3P) with
phosphoenolpyruvate (PEP) to form
5-enolpyruvylshikimate-3-phosphate (EPSP).
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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
• Complex, composed of 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).
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Insecticide Usage on Bt and non-Bt Cotton for
1999-2001
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Vaccine plants

• Pioneered by Charlie Arntzen
• cheap vaccine-delivery system
• use plants producing a pathogen protein (or DNA)
  to induce immunity
• 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.
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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