Making Transgenic Plants and

Animals
• Why?
1. Study gene function and regulation
2. Making new organismic tools for other fields
of research
3. Curing genetic diseases
4. Improving agriculture and related raw
materials
5. New sources of bioengineered drugs (use
plants instead of animals or bacteria)
 Genetic Engineering of Plants
• Must get DNA:
1. into the cells
2. integrated into the genome (unless using transient
expression assays)
3. expressed (everywhere or controlled)

• For (1) and (2), two main approaches for plants:

1. Agrobacterium - mediated gene transfer
2. 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.
 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
 Crown galls
caused by A.
tumefaciens on
nightshade.

More about Galls:

http://waynesword.palomar.edu/pljuly99.htm
http://kaweahoaks.com/html/galls_ofthe_voaks.
html
 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
Agrobacterium tumefaciens
 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
 Tumor characteristics

1. Synthesize a unique amino acid, called “opine”

– octopine and nopaline - derived from
arginine
– agropine - derived from glutamate
2. Opine depends on the strain of A. tumefaciens
3. Opines are catabolized by the bacteria, which
can use only the specific opine that it causes
the plant to produce.
4. Has obvious advantages for the bacteria, what
about the plant?
 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.
 Ti Plasmid
1. Large (200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– enzymes involved in opine metabolism
– proteins involved in mobilizing T-DNA (Vir
genes)
 T-DNA

LB auxA auxB cyt ocs 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!

 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.

 Vir (virulent) genes

1. On the Ti plasmid
2. Transfer the T-DNA to plant cell
3. Acetosyringone (AS) (a flavonoid) released by
wounded plant cells activates vir genes.
4. virA,B,C,D,E,F,G (7 complementation
groups, but some have multiple ORFs),
span about 30 kb of Ti plasmid.
 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
From Covey & Grierson
 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. 278:25273-25276

VirE2 may get DNA-protein complex across host PM

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

• 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
 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.
Binary vector system
2 Common Transformation Protocols

1. Leaf-disc transformation - after selection and

regeneration with tissue culture, get plants
with the introduced gene in every cell

2. 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, 735–743)
 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.
 Direct DNA transfer

• Introduce naked DNA into cells; assay

expression immediately or select for
permanently transformed cells

• DNA introduction:
1. Chemical
2. Electroporation
3. Particle bombardment (Biolistics)
 Chemically-Induced Transformation

• Usually use on cells without walls

• Multiple protocols (examples):
1. Put DNA inside artificial membranes
(liposomes), they will fuse with plasma
membrane.
2. Bind DNA with polycations to neutralize
charge, some cells endocytose the
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 or
diffusion (?)
• Drawback - its more
cumbersome to regenerate
plants from single
protoplasts than from the
tissue transformations with
Agrobacterium
 Particle Bombardment
• Less limitations than electroporation
• Can use on cells with walls, essentially any
tissue
• Can transform organelles!
• Method:
1. Precipitate DNA onto small tungsten or gold
particles.
2. Accelerate particles to high speeds at cells or
tissues.
3. Selective growth and regeneration of transgenic
plants as described for Agro-mediated
transformation.
 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

 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 or About to
be 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)
• Contain bacterial EPSP synthase
• Advantages: better weed control, less
tillage
• soybeans, corn, rice, wheat
The function of EPSP synthase is to
combine the substrate shikimate-3-
phosphate (S3P) with
phosphoenolpyruvate (PEP) to form 5-
enolpyruvylshikimate-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
– Complex, composed of several
proteins
– Cry and Cyt genes A Transmission
– encoded on a plasmid Electron Micrograph
• Advantage: less insecticide of negatively stained
required, better yield spores from Bt2-56
• corn, cotton, potatoes containing a filament
(a), and a sac-like
structure containing
a spore (b) and
parasporal body (c).
Insecticide Usage on Bt and non-Bt Cotton for 1999-2001
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.
 Concerns that have been raised about
cultivating and consuming GM crops

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 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