PLANT GENETIC ENGINEERING

twoeggfinnishBiotechnology

Dec 14, 2012 (4 years and 7 months ago)

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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 exist

to deliver vitamin pills



Improved vitamin A content in widely consumed

crops an attractive alternative


-
Carotene Pathway Problem in Plants

IPP

Geranylgeranyl

diphosphate

Phytoene

Lycopene



-
捡rot敮e

(v楴im楮iA⁰r散畲獯爩

Phytoene synthase

Phytoene desaturase

Lycopene
-
beta
-
cyclase

ξ
-
carotene desaturase


Problem:

Rice lacks

these enzymes

Normal

Vitamin A

“Deficient”

Rice

The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene



-
捡rot敮e

(v楴im楮iA⁰r散畲獯爩

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 A

Pathway

is complete

and functional

Golden

Rice

What are the Uses of GM Plants?


Bioreactors / Molecular farming


Therapeutic proteins


Human
lactoferrin

to treat iron deficiencies


Antibodies


Vaccine production


Antigen expression


HepC
, HIV

Antibody
-
producing tomato plant

Nicholas Ewing

California 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

11

Cloning vector development based on T
-
DNA

1.
Co
-
integrative vector

2.
Binary cloning vector

Cointegrate
vector

Disarmed
Ti
-
plasmid

Recombinant
Ti
-
plasmid

Co
-
integrative and binary vectors

Binary vector

t
-
DNA

VIR genes

Plasmid

DNA

Bacterial

Chromosome

Bacterial ORI

Ampicillin

resistance

LB

RB

Co
-
integrative

TRANSFORMATION

Metode

transformasi

pada

GM plant:

1.
Co
-
cultivation

2.
Electroporation

3.
Biolistic

transformation


“Gene gun”

Co
-
cultivation


Agrobacterium

contains
Ti plasmid

recombinant



Co
-
cultivation of the
Agrobacterium

with
plant pieces transfers
the DNA

Bacterial

chromosome

Ti Plasmid

Petri dish

with leaf pieces

plus
Agrobacterium


General transformation protocol

Agrobacterium

sp

Sterile explants

with dividing cells

Inoculate (mins
-
hrs)

(bacterial attachment)

Co
-
cultivate (days)

Transfer of t
-
DNA

Wash

Transfer to medium

with bactericidal

antibiotics (days)

Kill off Agrobacterium

Transfer to medium

with bactericidal

antibiotics plus

selective antibiotics

(months)

Kill off
Agrobacterium


and select transgenic

cells

Transfer to

regeneration

medium plus

selective

antibiotics

Regeneration

of transgenic

plants

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

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

39

39

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

Microinjection

40

Male pronuclei

Pregnant mare serum
gonadotropin


follicular

Human chorionic
gonadotropin

ovulation

40

41

41

A.Yuswanto, Fac. of Pharmacy, UGM

Retroviral infection

42

Drawback ?

42

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

Embryonic stem (ES) cell transfer

43

43

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

45

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