Metabolism and NutritionMetabolism and Nutrition 20122012

Genetically Modified OrganismsGenetically Modified Organisms (GMOs)(GMOs)

สิ่��งมี�ชี�วิ�ตสิ่��งมี�ชี�วิ�ตดั�ดัแปลงดั�ดัแปลง((แต�งแต�ง)) พั�นธุ์�� พั�นธุ์�� ((จี�เอ็�มีโอ็จี�เอ็�มีโอ็) )

(Prof. Dr. Jerapan Krungkrai)(Prof. Dr. Jerapan Krungkrai)

Metabolism and NutritionMetabolism and Nutrition 20122012

Genetically Modified OrganismsGenetically Modified Organisms (GMOs)(GMOs)

สิ่��งมี�ชี�วิ�ตสิ่��งมี�ชี�วิ�ตดั�ดัแปลงดั�ดัแปลง((แต�งแต�ง)) พั�นธุ์�� พั�นธุ์�� ((จี�เอ็�มีโอ็จี�เอ็�มีโอ็) )

(Prof. Dr. Jerapan Krungkrai)(Prof. Dr. Jerapan Krungkrai)

(1999 data)

Genetically Modified Organisms (GMOs) (จี�เอ็�มีโอ็)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.

- 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

1.1. Here, we concern only plant GMOs or GM crops

1.2. Modification Objectives

Agronomic trait- Drought resistance- Herbicide or salt tolerance- Nitrate reduction - Temperature resistance

Pest resistance- Bacterial resistance - Fungal resistance - Insect resistance - Nematode resistance - Viral resistance

1.3. Structure of a GMO

Promoter Coding region Terminator

e.g. Cauliflower Mosaic Virus promoter (CaMV 35S; p-35S)

e.g. Agrobacterium tumefaciens nopalin synthase terminator (t-NOS)

or t-35S

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 Terminator Altered trait

Maximizer®

(Bt-176, Bt-11)Corn Syngenta p-35S CryIA endotoxin

(Bacillusthuringiensis)Bar (Streptomyceshygroscopicus)

t-35S Insectresistance

Herbicidetolerance

Lyberty Link®

T-25Corn Aventis p-35S Pat (Streptomyces

viridochromogenes)t-35S Herbicide

tolerance

YieldGard®

MON810Corn Monsanto p-35S CryIA endotoxin

(Bacillusthuringiensis)CP4 epsps(Agrobacterium)

t-NOS Insectresistance

Herbicidetolerance

RoundupReady®

Soy beanCotton

Monsanto p-35S CP4 epsps(Agrobacterium)

t-NOS Herbicidetolerance

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.

- 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

Polygalacturonase << (reverse 3’5’)

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

(100 ha = 1 km2)

In 2009, 135 million hectares of GM crops planted in 25

countries. And ~ 63% of maize crop are GM.??

(Data 1999)

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. GMO construction

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 T-DNA 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

- Protein detection: Immunoassay- DNA detection: Conventional and Real-Time PCR

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.

7.1. Principle of detection of GMOs in foods

CaMV 35S Promoter

Coding region NOS Terminator

KanamycinMarker(npt II)

(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)

Lateral Flow strip

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.

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

- 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

7.4. GMOs Real-Time PCR using Taqman Probes

Promoter Coding region Terminator

Screening Assay

Specific Assay

Design of Screening and Specific Real-Time PCR Assays

Extraction reagent

20mg of sample

100100ºC 10 minutes10 minutes

Centrifugation

Supernatant for PCR

Sample processing:

Types: solid/granule, solid, liquid,viscous liquid

- 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

7.5. GMOs Reference Standards

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 anti-nutrient 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 commercial- ization 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 anti- cancer 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.

Good Luck and Have a Good Examination

Good Luck and Have a Good Examination

Evaluation: MCQs, 6 questions/5 choices

Genetic Engineering (Recombinant DNA Technology)

- Prof.Dr.Jerapan Krungkrai

Metabolism and Nutrition 2012Metabolism and Nutrition 2012

Reference: Alberts, B. et al. (2000 ) Mo lecular Biology of the Cell

Tobacco histone tagged with gre en fluorescent protein (GFP)

Techniques used in GMOs

• Cloning of gene

• PCR

Basic and Principle of Genetic Engineering

1. Cloning

Clone

Gene

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 andmaintaining 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

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.

Genetic engineering of plants is

much easier than that of animals:

Transgenic plants

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.

Ti based Plasmid

- Vir region codes for proteins helping in transferring of T-region

Hostspecific

ityregion

Virulenceregion

T-DNA

Ti plasmid

TransfectionTransfectionMethods-Electroporation-Microinjection-Biolistics (particle gene gun, particle bombardment)

* Plant cells has to be prepared as PROTOPLAST

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

Firing pin

Charge

Macroprojectile

Microprojectiles

Target cells

* Plant cells has to be prepared as PROTOPLAST

Gene Gun

Transform intoA. tumefaciens

Plant protoplast culture

inoculation plating

selection

Cloning of a plant gene

Natural transformation

Direct selectionGenetic Complementation

Drug resistance property (i.e. Kanamycin)

Auxotroph complementationMarker inactivation

Blue-white coloniesDrug sensitivity

How to select a clone of interest?

Direct expressed protein screening

1 2

culturingcandidate

colonies

cell extraction

1 2

1 2

protein analysis onto SDS PAGE

candidateband

expression libraries

molecular weight marker control cell lysate

Human Cloning statement

2. PCR (Polymerase Chain Reaction)

Principle of PCR

PCR, template and primers

DNA amplification by PCR

PCR product

Cycle number

15 18 21 30

30 ng

15 ng

10 ng

End-point

Conventional PCR - QualitativeReal-time PCR - Qualitative & Quantitative

DNA Target Amplification (Amplicon) = 2n, n = cycle number

No. of No. of AmpliconCycles Copies of Target

1 2

2 4

3 8

4 16

5 32

6 64

20 1,048,576

30 1,073,741,824

1 cycle = 2 Amplicon

2 cycle = 4 Amplicon

3 cycle = 8 Amplicon

4 cycle = 16 Amplicon

5 cycle = 32 Amplicon

6 cycle = 64 Amplicon

7 cycle = 128 Amplicon