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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)