PLANT GENETIC ENGINEERING

Agustina Setiawati

What are uses GM Plant?

Nutraceutical Golden rice

Vitamin A enriched

The Golden Rice Story

• Vitamin A deficiency is a major health problem• Causes blindness• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t existto deliver vitamin pills

• Improved vitamin A content in widely consumed crops an attractive alternative

-Carotene Pathway Problem in PlantsIPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A

“Deficient”Rice

The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

What are the Uses of GM Plants?

Bioreactors / Molecular farming Therapeutic proteins

Human lactoferrin to treat iron deficiencies Antibodies

Vaccine production Antigen expression

HepC, HIVAntibody-producing tomato plantNicholas EwingCalifornia State University, Sacramento

Agrobacterium sp Gram negative soil borne bacterium Causes crown gall tumours Mempunyai plasmid Ti yang bisa dipindahkan ke sel

tanaman

Crown-gall disease

A.tumefaciens

Hairy-root disease

A.rhizogenes

Plasmid Ti

The production of phytohormon prevent being regenerated into mature plants Ti-plasmid are large (200 to 800 kb) Ti-plasmid does not replicate in E.coli

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Cloning vector development based on T-DNA1. Co-integrative vector2. Binary cloning vector

Cointegrate vector

Disarmed Ti-plasmid

Recombinant Ti-plasmid

Co-integrative and binary vectors

Binary vector

t-DNA

VIR genesPlasmid DNA

BacterialChromosome

Bacterial ORIAmpicillin resistance

LB RB

Co-integrative

TRANSFORMATION

Metode transformasi pada GM plant:1. Co-cultivation2. Electroporation3. Biolistic transformation – “Gene

gun”

Co-cultivation

Agrobacterium contains Ti plasmid recombinant

Co-cultivation of the Agrobacterium with plant pieces transfers the DNA

Bacterial chromosomeTi Plasmid

Petri dishwith leaf pieces

plus Agrobacterium

General transformation protocolAgrobacterium sp

Sterile explantswith dividing cells

Inoculate (mins-hrs)(bacterial attachment)

Co-cultivate (days)Transfer of t-DNA

Wash

Transfer to mediumwith bactericidalantibiotics (days)Kill off Agrobacterium

Transfer to mediumwith bactericidalantibiotics plusselective antibiotics(months)Kill off Agrobacterium and select transgeniccells

Transfer to regenerationmedium plusselective antibioticsRegenerationof transgenicplants

Transformation

Recovery of transgenic plants

Electroporation Prinsip: pembukaan membran pembentukan pori

sel tanaman dengan muatan listrik DNA in the surrounding solution can enter the

cell through these pores and become incorporated into the cell’s nuclear genome through illegitimate recombination

Biolistic transformation – “Gene gun”

DNA is precipitated on the surface of heavy metal (gold; tungsten) particles

Loaded particles are driven into plant cells by high velocity gas propulsion (originally gunpowder; now helium)

Distance between discharge nozzle and tissue can be optimized, as can particle velocity

Target tissue must be regenerable

23 A.Yuswanto, Fac. of Pharmacy, UGM

EDIBLE VACCINE VIRUS-RESISTENT PLANT

GM Plant is used for?

EDIBLE VACCINE

Edible vaccines are vaccines produced in plants that can be administered directly through the ingestion of plant materials containing the vaccine. Eating the plant would then confer immunity against diseases.Edible vaccines produced by transgenic plants are attractive for many reasons. The cost associated with the production of the vaccine is low, especially since the vaccine can be ingested directly, and vaccine production can be rapidly up scaled should the need arises. Edible vaccine is likely to reach more individuals in developing countries. The first human clinical trial took place in 1997. Vaccine against the toxin from the bacteria E.coli was produced in potato. Ingestion of this transgenic potato resulted in satisfactory vaccinations and no adverse effects.

What exactly are “edible vaccines?”

• Biopharmaceuticals• Plants or crops that produce human vaccines• The next generation of vaccines

VIRUS RESISTANT PLANT

Transgenic Animals

SOMATIC NUCLEAR CELL TRANSFER

Transgenic animal was constructed based on SNCT by Robert et al (1952)

DOLLY SHEEP, first succeed cloning

Uses for transgenic animals

Gene pharming

Xenotransplantation

Industrial

Food/Feed

The foreign gene is constructed using recombinant DNA technology.

In addition to a structural gene, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host, and to be expressed correctly by the cells of the host.

Transgenic Animal

Recombinant protein production in the milk of a transgenic sheep

Knock-in A new gene is added (knocked in) by random

insertion into the genome of the host organism. Your goal is to express that gene, but you don’t care where it ends up in the genome. Circular plasmid construct DNA can break anywhere in its

sequence and insert anywhere into the recipient cell genome. Usually performed by microinjection into one of the

two pronuclei of a newly fertilized egg. Example:

Knock in a β-galactosidase gene driven by a promoter being tested for tissue specificity. Watch where and when the β-galactosidase is expressed.

TRANSGENIC ANIMALS

“A little bit of this, and a little bit of that?”

Methods of producing transgenic animal

1. DNA microinjection

2. Embryonic stem (ES) cell transfer

3. Retroviral infection

3939

Source: A.Yuswanto, Fac. of Pharmacy, UGM

Microinjection

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Male pronuclei

Pregnant mare serum gonadotropin follicularHuman chorionic gonadotropin ovulation40

4141 A.Yuswanto, Fac. of Pharmacy, UGM

Retroviral infection

42

Drawback ?

42 Source: A.Yuswanto, Fac. of Pharmacy, UGM

Embryonic stem (ES) cell transfer

4343 Source: A.Yuswanto, Fac. of Pharmacy, UGM

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Pronuclear injection.

High efficiency for random knock-ins.

How to analyse transgenic mice

Knock-out transgenic

A gene of the host organism is inactivated (knocked out) by insertion of a foreign sequence.

A mutant allele replaces the normal one by homologous recombination.

This is known as “targeted” insertion of a gene. Targeting involves incorporating sequence identical to

the target gene in the vector. Successful homologous recombination is rare and must

be Selected and Screened

KNOCKOUT MICE

Isolate gene X and insert it into vector.

Inactivate the gene by inserting a marker gene

that make cell resistent to antibiotic (e.g.

puromycin)Transfer vector with (-) gene X into ES cells

(embryonic stem)

MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

e.g.(NeoR)

TERIMA KASIH