Metabolism and Nutrition 2012
Genetically Modified Organisms (GMOs)
สิ่ งมีชีวติ ดัดแปลง(แต่ ง)พันธุ์ (จีเอ็มม อ็)
(Prof. Dr. Jerapan Krungkrai)
(1999 data)
Genetically Modified Organisms (GMOs)
(จีเอ็มม อ็)
Objectives: After this topic, the students should be able to:
1. Describe GMOs and examples of GMOs in our daily life.
2. Describe construction of GMOs using genetic engineering techniques.
3. Discuss safety and regulation of GMOs (environment and public health).
4. Describe detection and identification of GMO products.
Contents:
1. Definition of GMOs and examples of GMOs.
2. Construction of GMOs using genectic engineering.
3. Safety of GMOs in view of environment and public health aspects.
4. Regulation of GMOs by labeling and/or percentage of GMOs in food products.
5. Detection techniques and identification of GMO products.
6. Future trends of GMOs.
Teaching methods: 1-1.5 h lecture with power-points and hand-out sheets provided with
genetic engineering introduction.
Evaluations: 6 MCQs with 5 choices (including genetic engineering introduction)ใ
References: lists in the provided hand-out power-point sheets.
1. What are GMOs?
- Genetically Modified Organisms (GMOs)
are living organisms created through genetic
engineering. This has been described earlier.
- Scientists transplant the genes of one species
into another species to try to transfer
"desirable" characteristics.
1.1. Here, we concern only plant GMOs
or GM crops
- Plants containing stably-integrated exogenous
DNA encoding:
-
Herbicide resistance
Insect resistance
Reporter
Others
- Created using modern molecular biology
techniques rather than traditional selective
breeding
- Biolistic particle bombardment (gene gun)
- Agrobacterium tumefaciens transformation
1.2. Modification Objectives
Agronomic trait
Pest resistance
- Drought resistance
- Bacterial resistance
- Herbicide or salt tolerance
- Nitrate reduction
- Temperature resistance
- Fungal resistance
- Insect resistance
- Nematode resistance
- Viral resistance
1.3. Structure of a GMO
e.g. Cauliflower Mosaic Virus
promoter (CaMV 35S; p-35S)
e.g. Agrobacterium tumefaciens
nopalin synthase terminator (t-NOS)
or t-35S
Promoter
Coding region
Terminator
The CaMV 35S promoter is a strong constitutive (not regulated)
promoter used to drive expression of foreign genes in plants
Almost all transgenic plants approved for agricultural use rely on the
CaMV 35S promoter (p-35S)
1.4. Examples of GMOs
Product
Organism Producer Promoter Transgene
Maximizer®
(Bt-176, Bt-11)
Corn
Syngenta
p-35S
Lyberty Link®
T-25
Corn
Aventis
p-35S
YieldGard®
MON810
Corn
Monsanto p-35S
Roundup
Ready®
Soy bean
Cotton
Monsanto p-35S
Terminator Altered trait
CryIA endotoxin
t-35S
(Bacillus
thuringiensis)
Bar (Streptomyces
hygroscopicus)
Pat (Streptomyces t-35S
viridochromogenes)
CryIA endotoxin
(Bacillus
thuringiensis)
CP4 epsps
(Agrobacterium)
CP4 epsps
(Agrobacterium)
 Insect
resistance
 Herbicide
tolerance
t-NOS
 Herbicide
tolerance
 Insect
resistance
 Herbicide
tolerance
t-NOS
 Herbicide
tolerance
Pat = phosphinotricin (P=herbicide, glyfosinate)-N- acetyltransferase (= Bar, bialaphos
resistance).
Epsps = 5-enolpyruvylshikimate-3-phosphate synthase  for aromatic amino acids
synthesis;
Herbicide Roundup = glyphosate inhibit s epsps enzyme.
1.4. Examples of GMOs (cont’)
More GMOs:
•
•
•
•
•
•
•
•
tomato (ripening slower),
potato(insect resistance),
corn (male sterility),
squash (virus resistance), melon (virus resistance),
papaya (virus resistance),
rape seed or canola (high level of lauric acid),
tobacco (herbicide tolerance),
……………...
1.5. GM crops: History
- The first genetically improved crop was the Flavr Savr™ tomato
(Calgene company) which was approved in 1994, USA.
- 1983 Plant Genetic Engineering
Technology
- 1990 Recombinant chymosin (from
-
fungus Aspergillus niger of
bovine gene) replaces rennet
(from bovine intestine) enzyme
used in cheese production,
US FDA approved.
Polygalacturonase <<
(reverse 3’5’)
- 1994 Flavr Savr tomato approved in
USA
- 1995 Genetically modified soybeans
introduced on the market
- 1997 20 GM crops approved in US
- 1999 Golden Rice developed
Golden rice adds 3 enzymes for beta carotene synthesis from geranyl geranyl diphosphate
= phytoene synthase, phytoene desaturase, lycopene beta-cyclase.
1.6. GM crops: area production
In 2009, 135 million hectares of
GM crops planted in 25
countries. And ~ 63% of maize
crop are GM.??
(Data
1999)
(100 ha = 1 km2)
2. GMO construction
2.1. Plant Transformation Systems:
Monocot plants are usually transformed using a
biolistic particle bombardment or “gene gun”
Dicot plants are usually transformed using
Agrobacterium tumefaciens
Both systems require a mechanism for selection of
transformants and a plant promoter to drive
heterologous gene expression
2.1.1. Gene gun technique
2.1.2. Agrobacteria transformation technique
Agrobacterium tumefaciens is
a naturally occuring bacteria
that infects dicot plants
producing Crown gall disease
DNA sequences from the
agrobacteria are integrated
into the plant genome
Scientists have placed foreign
DNA into the agrobacteria for
selection and expression of
the foreign genes
2.2. Selection using marker
An antibiotic
resistance gene
(usually kanamycin
resistance) is
inserted into the TDNA plasmid
Plant tissue grown in
the presence of
kanamycin will not
grow due to loss of
chloroplast functions
Transformed tissue
can be grown into an
adult plant
3. Genetically Modified Crops: Example
 Numerous crop plants have been genetically
engineered for tolerance to the herbicide Roundup
An insecticidal protein from Bacillus thuringiensis
has been cloned and expressed in corn, soybean,
and cotton plants, namely Bt crops
B. thuringiensis is a bacteria that naturally
produces proteins toxic to certain insects (B.t.
toxin= CrylA endotoxin))
B.t. toxin produces channels in the membranes of
the gut in insects
B.t. toxin is not harmful to animals or humans
B.t. Toxin comments & B.t. cotton
Are B.t. crops protected from insect pests?
Do B.t. crops increase production yields?
Do farmers use less insecticides with B.t. crops?
Are B.t. crops harmful to insects not considered pests
(butterfly)?
Do insects become resistant to B.t. toxin?
Are B.t. crops harmful to the environment and biodiversity?
4. Environmental and public health safety
issues of the introduced genes in GM crops
- 1. Potential gene flow to other organisms:
bacteria, plants, mammals
- 2. Destruction of agricultural diversity
- 3. Allergenicity & toxicity
- 4. Antibiotic resistance
- 5. Gastrointestinal problems/nutritional
changes/anti-nutrient effect
5. Questions About GM Foods
How are plants genetically engineered?
What foreign DNA sequences are used in genetic
engineering and why?
How are crop plants modified and why?
What are the safety, social, ethical, and environmental
issues of GM foods?
How are GM foods regulated (US and worldwide)?
6. Labeling of GM Food Products
Should products derived from GM crops be
labeled as such?
What tests are currently used to detect GM
ingredients in food products?
What is an acceptable level of GM ingredients
in food products?
What is the current labeling policy (US and
worldwide)?
Labeling Regulations
- USA
- GMOs must be labeled.
- Switzerland
- Establishes 1% threshold for labeling effective July 1, 1999
- European Union
- EC/1139/1998 requires labeling of foods containing GM
ingredients, especially soy and maize
- EC/49/2000 establishes 1% labeling thresholds
- EC/1829/2003 establishes 0.9% labeling thresholds for
authorized GMOs and 0.5% for non-authorized GMOs in EC
- EC/1830/2003 requires traceability and labeling of GMOs
throughout the entire food and feed chain
- Japan
- Establishes 5% labeling threshold on April 1, 2000 based
upon major ingredients containing GMO and enforcement
effective April 1, 2001
7. GMOs Detections/Tests/Assays
7.1. Principle of detection of GMOs in foods
GM products contain an additional trait encoded
by an introduced gene(s),
which generally produce an additional protein(s)
that confers the trait of interest.
- Protein detection: Immunoassay
- DNA detection: Conventional and Real-Time PCR
Kanamycin
Marker
(npt II)
CaMV 35S
Promoter
Coding region
NOS
Terminator
(npt II= neomycin phosphotransferase)
7.2. GMOs Immunoassay
-
Fast and inexpensive (dipsticks)
Antibodies detect expressed protein
Qualitative, not quantitative
Only detects gene products expressed in test
material (tissue-specific expression)
- Sensitivity and specificity dependent upon
antibody
- Protein degradation during sample extraction can
limit detection
- Negative result does not indicate absence of
transgene (false negative result)
Solid phase immunoassay:
1) a competitive assay in which the detector and analyte
compete to bind with capture antibodies,
2) a two-site (double antibody sandwich) assay in which the
analyte is sandwiched between the capture antibody and the
detector antibody.
Lateral Flow strip
7.3. GMOs conventional PCR
-
Semi-quantitative endpoint analysis
Target present in all tissues
Potential for carry-over contamination
Sensitivity dependent upon detection method
Gel electrophoresis-based analysis
Labor-intensive, especially competitive PCR
Low throughput
Negative result does not indicate absence of
transgene (false negative result)
PCR Primers: p-35S or t-NOS
7.4. GMOs Real-Time PCR using Taqman Probes
-
Quantitative results
Greater specificity provided by TaqMan probe
No carry-over contamination
Integrated and automated amplification, detection,
data collection and analysis
- Gel-free analysis
- High throughput, 96- and 384-well formats
- Prevention of false negative result
Screening Assay
Promoter
Coding region
Terminator
Specific Assay
Design of Screening and Specific Real-Time PCR Assays
Sample processing:
Types: solid/granule, solid, liquid,viscous liquid
20mg of sample
100ºC
10 minutes
Extraction reagent
Centrifugation
Supernatant
for PCR
7.5. GMOs Reference Standards
- Manufactured by the Institute of Reference
Materials and Measurements, IRMM (Geel,
Belgium)
- GMO flour blended with non-GMO flour to
obtain specific % GMO content
- Soy and Maize reference standards available
- Provide quantitation standard and DNA template
preparation control
7.6. GMOs Real-Time PCR/ %GMO detection
Example: 1. Automated Quantitation of Real-Time PCR
2. Results from GMO positive soy sample (Energy Bar)
CaMV 35S amplification plot for Energy Bar sample.
Calculated %GM = 11%
8. GM crop safety evaluation strategies
- Protein safety evaluation:
In addition to acute toxicity, the other main
adverse effects associated with proteins are antinutrient effects (e.g. soybean trypsin inhibitors),
effects on the immune system (e.g. lectins) and
allergenicity (e.g. soybean)
- Requirement for animal studies:
If the characterization of the food indicates that
the available data are insufficient for a thorough
safety assessment, animal testing may be deemed
necessary.
9.Thailand and GM crops
•1. Chili
•2. Tomato
•3. Rice (Stunt virus)
•4. Papaya (Ringspot virus, not from
Cornell university)
•5. Cotton (Thailand native lines, not
from Monsanto company)
•6. Pineapple
In Thailand, we concern GM crops
and foods since 1993, however, in 1999
there are regulations for the GMOs.
“ GM crops productions for commercialization are prohibited, only research in
laboratory /controlled area can be studied.”
10. Future Trends of GMOs
Up to 2010, more than 48 GM crops will be developed !!!
1. Agronomical quality: slower ripening (banana), herbicide
tolerance (wheat, sunflower….), virus and fungus
resistance (rice, melon, cucumber…)……
2. Nutritional quality: richer in starch (potato), high level
of amino acids (soy bean), lower level of saturated fats
in oil (corn)……
3. Pharmaceutical quality: higher level of a natural anticancer agent (strawberry), producing a vaccine against
hepatitis B virus (banana), producing antioxidant agents
(Broccoli), synthesizing hemoglobin (tobacco)…….
4. Industrial quality: colored fibers (cotton)……..
5. Transgenic animals (pigs) expressing plant genes coding
for PUFAs.
References
1.Gachet, E. et al. (1998) Trends in Food Science & Technology 9, 380-388.
2. Beachy, R.E. (1999) Science 285, 335.
3. Doerfler, W. et al. (2001) Annals of the New York Academy of Sciences 945, 276-288.
4. Ahmed, F.A. (2002) Trends in Biotechnology 20, 215-223.
5. Nap, J.P. et al. (2003) Plant Journal 33, 1-18.
6. Corner, A.J. et al. (2003) Plant Journal 33, 19-46.
7. Taverniers, I. et al. (2004) Biotech International 16, 20-23.
8. Niemann, H. (2004) Proceedings of the National Academy of Sciences 101, 7211-7212.
9.Rosi-Marshall, E. et al. (2007) Proceedings of the National Academy of Sciences 104,
16204-16208.
10.Waltz E. (2009) Nature 461, 27-32.
11.Hutchison, W.D. et al. (2010) Science 330, 222-225.
12.Tabashnik, B. (2010) Science 330, 189-190.
Evaluation: MCQs, 6 questions/5 choices
Good Luck and
Have a Good Examination
Metabolism and Nutrition 2012
Genetic Engineering
(Recombinant DNA Technology)
- Prof.Dr.Jerapan Krungkrai
Techniques used in GMOs
•
Cloning of gene
•
PCR
Tobacco histone tagged with green fluorescent protein (GFP)
Reference: Alberts, B. et al. (2000) Molecular Biology of the Cell
Basic and Principle of Genetic Engineering
1. Cloning
Gene
Clone
Cloning and library
Cloning and expression
Basic requirements for gene cloning
1. DNA sample containing fragments of gene of interest
(PCR or RT-PCR technique)
2. Vector DNA for transporting the gene into and
maintaining them within the host cell.
(plasmid, virus etc.)
3. Host cells that allow the vector to enter, replicate
and/or express the interested gene into protein.
( bacteria, yeast, plant etc.)
-transformation
-transfection
Genetic engineering of plants is
much easier than that of animals:
1.
There is a natural transformation system for plants
(the bacterium Agrobacterium tumefaciens ).
2.
Plant tissue can redifferentiate (a transformed piece
of leaf may be regenerated to a whole plant).
3.
Plant transformation and regeneration are relatively
easy for a variety of plants.
Transgenic plants
Ti based Plasmid
Ti (tumor inducing) plasmid of A. tumefaciens,
(greater 200 Kb)
- contain T-DNA, 15-30 Kb in size.
- T-DNA contain genes responsible for
cancerous growth in plant and synthesizing
unusual compounds, called opines,
that the bacterium use as nutrient.
- Vir region codes for proteins helping in transferring
of T-region
Virulence
region
Ti plasmid
T-DNA
Host
specificity
region
Transfection
Methods
-Electroporation
-Microinjection
-Biolistics (particle gene gun, particle bombardment)
DNA-coated microscopic particles, called
microprojectiles, are fired into the cell using a special
gene gun.
Microprojectiles, typically 1 micron (m), can
penetrate the membrane with minimal damage.
: Tungsten particle Gold particle
* Plant cells has to be prepared as PROTOPLAST
Gene Gun
Firing pin
Charge
Macroprojectile
Microprojectiles
Target cells
* Plant cells has to be prepared as PROTOPLAST
Natural transformation
inoculation
plating
Transform into
A. tumefaciens
selection
Plant protoplast culture
Cloning of a plant gene
How to select a clone of interest?
Direct selection
Genetic Complementation
Drug resistance property (i.e. Kanamycin)
Auxotroph complementation
Marker inactivation
Blue-white colonies
Drug sensitivity
Direct expressed protein screening
expression libraries
protein analysis onto SDS PAGE
culturing
candidate
colonies
1
1
2
1
2
2
cell
extraction
molecular weight
marker
candidate
band
control cell lysate
Human Cloning statement
2. PCR (Polymerase Chain Reaction)
Principle of PCR
PCR, template and primers
DNA amplification by PCR
Conventional PCR - Qualitative
Real-time PCR
- Qualitative & Quantitative
PCR product
End-point
30 ng
15 ng
10 ng
15
18
21
Cycle number
30
DNA Target Amplification (Amplicon) = 2n, n = cycle number
1 cycle = 2
Amplicon
No. of
Cycles
No. of Amplicon
Copies of Target
1
2
2
4
3
8
4
16
5
32
6
64
2 cycle = 4 Amplicon
3 cycle = 8
4 cycle = 16
5 cycle = 32
Amplicon
Amplicon
Amplicon
6 cycle = 64 Amplicon
7 cycle = 128
Amplicon
20
1,048,576
30
1,073,741,824