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Title: Recombinant DNA technology for genetically modifying organisms with examples of applications in plan


1
Recombinant DNA technology for genetically
modifying organisms with examples of applications
in plants
2
Why create genetically modified organisms?
  • Availability/accessibility of pharmaceutical
    proteins
  • vaccine antigens
  • human therapeutic proteins
  • Increasing global demand for quality food supply
  • improved production on the same acreage i.e.
    pest resistance...
  • ability to utilize available, marginal land
    i.e. saline soils, drought prone regions
  • Renewable energy resource, environmental
    remediation

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Cheese Please!
  • Rennin can be isolated from stomachs of
    slaughtered calf
  • Or produced in a bacteria as a GMO
  • About 60 of commercial cheese production uses
    GMO rennin

5
Genetically Modified Crops
  • Humans have been manipulating crops since the
    advent of agriculture

6
Commercial - U.S. GM Crops (percentage represents
share of overall crop that is GM)

Soybeans1 91 (2007)
Cotton1 87 (2007)
Canola2 55 (2001)
Papaya2 53 (2000)
Corn1 73 (2007)
1Source USDA, National Agriculture Statistical
Services June 2001 Acreage Report 2Pew
Initiative on Food and Biotechnology Factsheet,
August 2001
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Why Develop Transgenic Plants?
  • Improve value of crop plants
  • increased yield
  • increased environmental resistance
  • desirable ornamental properties
  • Bioreactors for protein/metabolite production
  • Basic research

Traditional plant breeding is slow, imprecise,
and restricted Transgenic plant a fertile
plant that carries an introduced gene(s) in its
germ line
9
How do you make a transgenic plant or GMO?
10
Gene Transfer Methods for Plants
Not monocots
? to organelles
Protoplasts
Methods of choice Ti plasmid-mediated gene
transfer Microprojectile bombardment
11
Vectors fundamental features similar for
different systems
  • Essential components of a plasmid
  • Origin of replication
  • eukaryotic replication (orieuk)
  • E. coli replication (oriE)
  • Drug selection markers
  • eukaryotic selectable marker (ESM)
  • prokaryotic selectable marker (Ampr)
  • Restriction enzyme sites
  • MCS multiple cloning site

Shuttle vectors Allow use in both prokaryotes and
eukaryotes Manipulation / propagation in
prokaryote Expression in eukaryote
p promoter t termination and polyadenylation
signals
12
Construct your transgene
13
Ti Plasmid-Derived Vector Systems
Lab-friendly vectors derived from Ti plasmid by
molecular biology
Features right border sequence multiple cloning
site plant selectable marker bacterial
selectable marker E. coli origin of replication
14
T-DNA is the vector
Agrobacterium-mediated transformation
15
Agrobacterium tumefaciens Genetic transformation
of plants
Plant injury Attachment at wound site Wound
response compounds released by plant Induction
of virulence genes On Ti plasmid DNA transfer
(T-DNA) to plant
A. tumefaciens
Crown gall tumor
16
Gene Gun
17
Selection of transformed plants the utility of
the marker gene
18
  • Regeneration of Transformed Plants
  • a difficult process

19
Nuclear Transgenic Plants
20
Herbicide-Resistant Plants
10 global crop loss to weeds 10 billion/year
herbicides Herbicides non-specific
need early application
persist in environment
Herbicide resistance Inhibit herbicide
uptake Overproduce targeted protein Prevent
binding to target protein Metabolic inactivation
of herbicide
21
Roundup Ready soybeans, without and with
Roundup (glyphosate) treatment
Source Monsanto
22
Delayed Flower Senescence
Alternative strategy Increased production of the
plant hormone cytokinin with the ipt gene
control
ipt
0 1 2
3 11
Days after pollination
Source Michelle Jones, Ohio State Univ.
23
In the interest of improved production of
pharmaceutical proteins and efficient production
of green energy, ask the question Can we
replace plants
With plants?
24
Plants as Bioreactors
Tobacco -vs- E. coli
E. Coli fermentation requires expensive equipment
and personnel Loss of plasmid from E. coli
cultures over time Modification and assembly of
(active) eukaryotic proteins often not possible
in E. coli 1 tobacco plant produces more
recombinant protein than a 300-liter E. coli
fermenter 50x cost to produce 1kg of a
recombinant protein in E. coli than in
transgenic plants
25
Plants as Bioreactors
Plants vs. Cultured Cells
Cultured cells susceptible to contamination Expe
nsive method of protein production 5,000 / gram
in culture cells 10-100 / gram
in plants Production in seeds for long-term
storage
Industrial scale requires efficient protein
purification
26
Plants as Bioreactors
Production of therapeutic agents in transgenic
plants
Done on a laboratory scale Industrial scale
requires efficient protein purification
27
Golden Rice
Nutritional enhancement
28
Manipulation of Nutritional Content
Vitamins
Rice Staple food for 3 billion people Deficient
in vitamin A 1-2 million deaths / year
blindness Golden Rice rich in vitamin
A Contains genes for b-carotene biosynthesis
pathway b-carotene generates gold color Excess
b-carotene has no harmful effects Strategy Ti
plasmid co-transformation psy and crt genes on
vector with no selectable marker lcy gene on
separate vector with selectable marker removal
selectable marker plant w/out antibiotic
resistance
Increased vitamin E content Overexpression of a
single gene in biosynthetic pathway
29
Insect Resistance Management
30
Insect-Resistant Plants Bt Toxins
Results of insect infestation on Bt (right) and
non-Bt (left) cotton bolls
Agricultural Research, 1991
31
Insect-Resistant Plants Bt Toxins
A new type of Bt corn for control of corn
rootworm was approved in 2003 It may result in
major reductions in soil-applied insecticides,
but brings new concerns for resistance
development.
Source USDA
Damage from corn rootworm feeding from severe
(left) to no damage (right)
32
Prevention of Bt-Resistant Insects
Combined Treatments
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Genetic Potential of the Chloroplast
  • Double stranded circular plasmid DNA
  • 120-160 Kb in a typical flowering plant, 130
    genes
  • A single leaf cell may have up to 100 (or more!)
    chloroplasts, each with up to 100 copies of the
    plasmid (10 000 genome copies/cell)

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Integration by Homologous Recombination
Left and right targeting regions segments of
plastid genome included in transformation vector
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AGRONOMIC TRAITS EXPRESSED VIA CHLOROPLAST
GENETIC ENGINEERING
46
Phytoremediation via the Chloroplast Genome
47
Antifungal or antibacterial activity by magainin
derivative MSI 99, a 20 aa cationic
antimicrobial peptide from frog skin.
48
Plants as Edible Vaccines
  • Inexpensive
  • No cold storage
  • No purification
  • No sterilization
  • No delivery system (needles)

Possible Delivery Foods Bananas Tomatoes Lettu
ce Carrots Peanuts Corn
Plant-derived antigen binds to M cells present in
lining of intestine Presented to macrophages /B
cells.
49
Are GMOs environmentally safe?
  • Yes, so far.
  • Numerous studies have searched for gene transfer
    to other organisms bacteria, plant, and insect
    with only one very specialized case of gene
    transfer.
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