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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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Recombinant Ti plasmid by recombination
Gene transfer by cointegration
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Microprojectile bombardment – “Shotgun”
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Viral Vectors
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Transfer into protoplasts
Gene transfer across a protoplast membrane is promoted by some chemicals such as polyethylene glycol
Vector + 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
- 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,..)
Manipulations that make a plant herbicide resistance
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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
Embryonic stem cells (ES)
Used for retroviral infection
Fertilized egg
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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
Inner cell mass (ICM) of blastocysts can form all cells of the embryo -> Pluripotent-> Embryonic stem cells
Engineered ES -> can form any kind of cell in an embryo
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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)
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.
Until 1997, arrival of Dolly – not possible to produce an adult animal from a nucleus from an adult animal´s differentiated cell
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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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Production of pharmaceutical proteins -> drugs
Problems:
Highly inefficient
Only 20% of the eggs survive and only 5% of them produce product
Transgenic Cattle, Sheep, Goat, Pigs
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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)