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Transcript
Transgenic Animals and
Plants
- Genetic Engineering of plant -> Transgenic plants
- Genetic Engineering of animals -> Transgenic animals
1
Definition of Transgenic
Transgenic -> stable introduction of a gene into another organism
-> For Unicellular organisms (such as bacteria or yeast)
all transformed cells are -> transgenic
-> For multicellular organisms (such as animals, plants,..)
difference between:
- manipulation of single cells -> cell line
(expression in insect cells or mammalian cells)
- manipulation of a whole plant or animal -> transgenic
(can have a transgenic offspring!!!)
-> more difficult and expensive to create whole modified organism
(transgenic) than just cell line!!!
2
Transgenic versus Cloning
Transgenic -> creation of transgenic animal or plant (introduction
of foreign gene into organism)
-> transgenic organisms produced by introduction of foreign gene into germ line
(-> transgenic offspring!!!)
-> introduction of gene into somatic cells -> gene therapy
Cloning -> obtaining an organism that is genetically identical to the
original organism
-> such as Dolly the sheep
-> asexual propagation of plants (taking cuttings)
3
Transgenic Plants
Why do we need transgenic plants ?
•
improvement of agricultural value of plant (resistance to herbicides,
resistance to insect attack -> Bacillus thuringiensis toxin)
•
living bioreactor -> produce specific proteins
•
studying action of genes during development or other biological
processes (knock-out plants, expression down-regulated)
4
Transgenic Plants
• Advantages:
- Plant cells are totipotent -> whole plant can be regenerated from
a single cell (engineered cells -> engineered plants)
- Plants have many offspring -> rare combinations and mutations
can be found
- Transposons used as vectors
• Disadvantages:
- Large genomes (polypoid -> presence of many genomes in one
cell)
- plants regenerating from single cells are not genetically
homogenous (genetically instable)
5
6
Gene – transfer methods
7
Agrobacterium tumefaciens mediated transfer
8
Ti Plasmid
9
Integration of T-DNA into the plant chromosome
-> Tumor formation
10
Gene transfer
by
cointegration
Recombinant Ti plasmid
by recombination
11
Microprojectile bombardment – “Shotgun”
12
Viral Vectors
13
Transfer into protoplasts
Vector + polyethylene
glycol
Gene transfer across a
protoplast membrane is
promoted by some chemicals
such as polyethylene glycol
14
Electroporation
15
Control elements on vector
Frequently used promoter: -> 35S promoter from cauliflower mosaic virus
16
Alterations in plant RNA – downregulation of specific genes
PG (polygalacturonase) -> Sensitivity
of tomatoes to bruising
Reduced level-> should give harder
fruit during shipping
Result: lower level -> did not give
harder fruit (more factors
responsible for process)
Expression of Antisense RNA of
transcript of PG -> reduces level of
protein produced
17
Selection marker free transgenic plant
-> Transposons
18
Applications for engineering plants
•
Development of Insect-, pathogen-, herbicide- resistant plants
•
Flower pigmentation
•
Modification of nutritional content
•
Modification of taste and appearance
•
Bioreactor
•
Vaccines (Cholera toxin-like protein in potatoes)
•
Plant yield (alteration of lignin content -> paper industry)
19
Development of Insect-, pathogen-,
herbicide- resistant plants
Toxin from Bacillus thuringiensis
20
Development of Insect-, pathogen-,
herbicide- resistant plants
21
Development of Insect-, pathogen-,
herbicide- resistant plants
Manipulations that make a plant herbicide resistance
- Inhibit the uptake of the herbicide
- overproduce the herbicide-sensitive target protein (Glyphosate)
- reduce ability of target protein to bind herbicide (cyclohexanediones)
- plant can degrade herbicide (Bromoxynil, Glufosinate, Cyanamide,..)
22
Development of Insect-, pathogen-,
herbicide- resistant plants
Fungus- and Bacterium- resistant plants
Engineering of plants
-> express
antimicrobial peptides
23
Flower pigmentation
CHS -> Chalone
synthetase -> enzyme in
biosynthetic pathway of a
purple pigment
24
Changed nutrition content
- Amino acids (to increase lysine content in the future in animal food)
- Lipids (possible to change degree of unsaturation, chain length)
- Vitamins (Vitamin E, increase Vitamin A in rice)
25
Modification of taste and appearance
Engineer potatoes -> produce more glucose and fructose at
higher temperatures
26
Plants as bioreactor
-Therapeutic agents
- Antibodies
- polymers (PHB)
27
Transgenic Animals
Transgene ->
Gene coding for a
growth hormone
28
Transgenic Animals
Why do we need transgenic animals ?
• living bioreactor -> produce specific proteins in the milk
(cattle, sheep, goats, pigs)
• studying action of genes during development or other biological
processes (knock-out animals, expression down-regulated) ->
models for studying human diseases -> mice
• improvement of agricultural value (fish, bird)
29
Gene-transfer methods
• Microinjection
• Retroviral method
• Engineered Embryonic Stem Cells (ES) method
• Knock – out methods (Cre-LoxP system) -> studying gene
expression + development
30
The first days of an embryo
Used for retroviral infection
Fertilized egg
Embryonic
stem cells
(ES)
31
Microinjection
into the germ line -> transgenic animal
Gene injected into the male pronuclei
32
Efficiency of the transgenesis process after
DNA microinjection
33
Retroviral vectors
into the germ line (8-cell embryo infected)
-> transgenic animal
34
Engineered Embryonic
Stem Cells (ES)
into the germ line (blastocyst)
-> transgenic animal
Engineered ES -> can form any kind of cell in an
embryo
Inner cell mass (ICM) of blastocysts can
form all cells of the embryo -> Pluripotent
-> Embryonic stem cells
35
Gene Therapy – Viral gene transfer into somatic
cells
Gene transfer into somatic stem cells -> gene therapy
Gene transfer via Virus
Target tissues: Bone marrow, liver, brain,....
36
Gene Therapy – Viral gene transfer into somatic
cells
Gene transfer into somatic stem cells -> gene therapy
Used for treating -> genetic diseases, such as diabetes, cancer, color blindness…
Different delivery methods
37
Gene Transfer - what happens on DNA level
Integration into chromosome -> Recombinantion
Recombinantion can be -> homologous – non-homologous
- non-homologous event -> more frequently
- homologous event -> less frequent but desired
Knock-out mutants -> disrupt functional gene by integration of
another gene into target gene
Used for:
-> study human diseases by creating model organisms
-> make minus mutant
38
Homologous recombinantion
39
How do check for homologous recombinantion
40
Construction of a disruption construct
41
42
43
44
Cre-LoxP system:
- Inactivation of a gene (knock-out) in a specific cell type
- Activation of a transgene in specific cell type
Used for:
- Study biological consequences of tissue- specific gene inactivation
-> establishing models for human diseases
-> selective removal of kinesin II gene (expressed in retinal receptor cells)
-> leads to accumulation of opsin and arrestin -> cell death
-> result mimics aspects of a disease (inherited retinis pigmentosa)
-> large deletions in chromosome -> deletion in chr. 22 -> DiGeorge syndrome
(cardiovascular dysfunction)
45
Inactivation of gene in specific cell type (tissue)
46
Cloning of Dolly – Cloning Animals by
Nuclear Transfer Technology
Critical for success:
Cell cycle of the somatic cells
(udder cells) on plates was critical –
they were kept in specific growth
stage (diploid stage)
Until 1997, arrival of
Dolly – not possible to
produce an adult
animal from a nucleus
from an adult
animal´s
differentiated cell
Of the 434 fused oocytes created
during the experiment -> only Dolly
survived to adulthood
Dolly was real clone (genotype
identical) and could reproduce
Dolly was euthanized 2003 ->
suffering from progressive lung
disease
Since 1997 -> cloning of sheep,
cows, mice, cats, other animals done
-> many of the clones developed
severe diseases as they matured.
47
Cloning of Mammals – Reproductive Cloning
- Genotype identical
- Phenotype is not necessarily identical -> variation due to
random events and due to environment
48
Why do clones have health problems?
Telomeres are found at the end of
each chromosome.
Shrinking of the telomeric ends of
our chromosomes are a sign of aging
of the cell.
Each cycle of cell division the
telomeres are slightly shortened
until they are too short for further
replication -> cell death
Dolly´s telomeres (at the age of 3)
have been as short as ones of the
age of 6 -> clones age “faster”.
49
Why do clones have health problems?
Differentiated cells have certain methylation pattern.
Cloned animals have abnormal methylation pattern originating from nucleus from
differentiated cells
Some can be “re-set” (epigenetic reprogramming) to their undifferentiated
state, some cannot -> faulty gene activation in cloned animal
-> so few cloned embryos survive
-> surviving clones have severe health problems
50
Transgenic Cattle, Sheep, Goat, Pigs
Production of
pharmaceutical
proteins -> drugs
Problems:
Highly inefficient
Only 20% of the
eggs survive and
only 5% of them
produce product
51
Transgenic Cattle, Sheep, Goat, Pigs
- Protein production: in milk, blood, urin
- Animals (pigs) with modification of sugars on surface of organs
-> donor for organ transplants
52
Transgenic Cattle, Sheep, Goat, Pigs
53
Transgenic birds and fish
-> improvement of agricultural value
Transgenic chicken:
- Resistant to viral, bacterial diseases
- better feeding efficiency (fast growth, better meat quality, more meat
- less fat meat, less cholesterol in eggs
- maybe use of eggs as bioreactors for protein production
Transgenic fish: -> to support aquaculture
- Increase growth rate (growth hormone)
- resistance to diseases
- Generation of model systems to monitor health hazard
(screening chemicals if they cause mutations)
54