Transgenic Animals and
Plants
- Genetic Engineering of plant -> Transgenic plants
- Genetic Engineering of animals -> Transgenic animals
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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!!!
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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)
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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)
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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)
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Gene – transfer methods
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Agrobacterium tumefaciens mediated transfer
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Ti Plasmid
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Integration of T-DNA into the plant chromosome
-> Tumor formation
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Gene transfer
by
cointegration
Recombinant Ti plasmid
by recombination
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Microprojectile bombardment – “Shotgun”
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Viral Vectors
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Transfer into protoplasts
Vector + polyethylene
glycol
Gene transfer across a
protoplast membrane is
promoted by some chemicals
such as polyethylene glycol
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Electroporation
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Control elements on vector
Frequently used promoter: -> 35S promoter from cauliflower mosaic virus
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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
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Selection marker free transgenic plant
-> Transposons
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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)
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Development of Insect-, pathogen-,
herbicide- resistant plants
Toxin from Bacillus thuringiensis
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Development of Insect-, pathogen-,
herbicide- resistant plants
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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,..)
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Development of Insect-, pathogen-,
herbicide- resistant plants
Fungus- and Bacterium- resistant plants
Engineering of plants
-> express
antimicrobial peptides
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Flower pigmentation
CHS -> Chalone
synthetase -> enzyme in
biosynthetic pathway of a
purple pigment
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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)
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Modification of taste and appearance
Engineer potatoes -> produce more glucose and fructose at
higher temperatures
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Plants as bioreactor
-Therapeutic agents
- Antibodies
- polymers (PHB)
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Transgenic Animals
Transgene ->
Gene coding for a
growth hormone
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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)
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Gene-transfer methods
• Microinjection
• Retroviral method
• Engineered Embryonic Stem Cells (ES) method
• Knock – out methods (Cre-LoxP system) -> studying gene
expression + development
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The first days of an embryo
Used for retroviral infection
Fertilized egg
Embryonic
stem cells
(ES)
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Microinjection
into the germ line -> transgenic animal
Gene injected into the male pronuclei
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Efficiency of the transgenesis process after
DNA microinjection
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Retroviral vectors
into the germ line (8-cell embryo infected)
-> transgenic animal
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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
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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,....
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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
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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
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Homologous recombinantion
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How do check for homologous recombinantion
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Construction of a disruption construct
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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)
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Inactivation of gene in specific cell type (tissue)
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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.
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Cloning of Mammals – Reproductive Cloning
- Genotype identical
- Phenotype is not necessarily identical -> variation due to
random events and due to environment
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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”.
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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
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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
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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
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Transgenic Cattle, Sheep, Goat, Pigs
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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)
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