Chapter 15: Genetically Modified Organisms: Use in Basic and Applied Research
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Transcript of Chapter 15: Genetically Modified Organisms: Use in Basic and Applied Research
Chapter 15:
Genetically Modified Organisms: Use in Basic
and Applied Research
Dolly is living proof that an adult cell can revert to embryonic stage and produce a full new being. This was not supposed to happen.
Charles Krauthammer, Time (1997) 149:60
15.1 Introduction
• Genetically modified organisms are no longer the realm of science fiction…
Transgenic organism
• Carries transferred genetic material (the transgene) that has been inserted into its genome at a random site.
Knockout organism
• Created by gene targeting—the replacement or mutation of a particular gene.
Cloned organism
• A genetically-identical organism produced by nuclear transfer from adult somatic (body) cells to an unfertilized egg.
15.2 Transgenic mice
• 1980: the first transgenic mouse was produced by microinjection of foreign DNA into fertilized eggs.
• 1982: “Super” mice expressing rat growth hormone gene coding sequence.
OncoMouse patent
• Is a transgenic mouse an invention?
• US patent for a mouse whose germ cells and somatic cells contain an activated oncogene sequence.
• The patent remains controversial worldwide.
How to make a transgenic mice
Three main stages in the process:
1.Microinjection of DNA into the pronucleus of a fertilized mouse egg.
2. Implantation of the microinjected embryo into a foster mother.
3.Analysis of mouse pups and subsequent generations for the stable integration and expression of the transgene.
Pronuclear microinjection
• Transgene: What are the minimal requirements for expression of a cDNA?
• Critical window of time before pronuclei fuse to form a diploid zygotic nucleus.
• Usually inject the sperm pronucleus since it is larger and closer to the egg surface.
Implantation into foster mother
• Manipulated embryos are transferred into a recipient “pseudopregnant” mouse.
• Pregnancy is visible about 2 weeks after embryo transfer.
• Litter is delivered about 1 week later.
Analysis of mouse pups
Two important questions:
• Is there stable integration of the transgene into the mouse chromosome.
• If the transgene is present, is it expressed appropriately?
Analysis of stable integration
• Success rate is ~2.5 to 6% in mice.
• Tail biopsies for DNA analysis by Southern blot or PCR.
• Integration is random and occurs by nonhomologous recombination.
• More than one copy may be integrated.
Analysis of transgene expression
At the level of transcription• Northern blots• RT-PCR• In situ hybridization, etc.
At the level of translation• Western blots• Immunohistochemistry• GFP expression, etc.
Transposon tagging
• Transposable elements have provided a powerful tool for insertional mutagenesis studies.
• A method to link phenotype with genomic sequence.
• Example: A transposon carrying antibiotic resistance is introduced into pathogenic bacteria.
• Screen for nonfunctional mutants, which indicates that insertion of the transposon disrupted a gene important for pathogenicity.
• Example: Gene knockout in mice by insertional mutagenesis using a “Sleeping Beauty” transposon.
• The mouse strain already contains the Sleeping Beauty transposase.
• Transposition activity is marked by activation of GFP at the new location.
Inducible transgenic mice
• What can be done if the transgenic is embryonic lethal?
• e.g. Inducible “Tet-off” expression system
15.3 Gene-targeted mouse models
• The ability to create a mouse of any desired genotype.
• A US-based consortium is systematically knocking out mouse genes one by one in embryonic stem cells.
• A European-based consortium is engineering knockout cells containing genes that can be switched on or off at any stage of development in the mutant mouse.
Knockout mice
Five main stages:
1.Construction of the targeting vector.
2.Gene targeting in embryo-derived stem (ES) cells.
3.Selection of gene-targeted ES cells.
4. Introduction of ES cells into mouse embryos and implantation into a foster mother.
5. Analysis of chimeric mice and inbreeding to obtain a pure breeding strain of “knockout mice.”
• The phenotype of the knockout mouse displays the impact of the targeted gene on development and physiology.
• Example: Argonaute2 knockout mice show severe developmental delay.
Knockin mice
• Often used for in vivo site-directed mutagenesis.
• Mutant knockin allele replaces the coding region of the endogenous allele.
Knockdown mice
• Analysis of cis-regulatory regions.
• Knockdown targeting sequence disrupts endogenous upstream regulatory elements, while keeping the coding region intact.
Conditional knockout and knockin mice
• Gene knockouts often result in embryonic lethality.
• To study a gene’s role later in development, genetic switches such as the Cre/lox system are used.
Cre/lox system for site-specific recombination
• Cre recognizes a 34 bp site on the bacteriophage P1 genome called lox.
• Catalyzes reciprocal recombination between pairs of lox sites.
Inducible gene expression in mice using the Cre/lox system
• Activation of transgene expression by site-specific recombination.
Conditional knockout by Cre-mediated recombination
• Modify the target gene in ES cells so that it is flanked by lox sites.
• Mice containing the modified gene are crossed with mice expressing Cre in the desired target tissue.
• Cre-mediated excision results in tissue-specific gene knockout.
15.4 Other applications of transgenic animal technology
• Transgenic animals have been explored as tools for applied purposes, ranging from artwork to pharmaceuticals.
Transgenic artwork: the GFP bunny
• Alba the GFP bunny was commissioned by artist Eduardo Kac.
Transgenic primates
• Mice do not always provide an accurate model of human physiology and disease pathology.
• Interest in extending transgenic and gene-targeting studies to nonhuman primates.
• 2001: ANDi, the first transgenic rhesus monkey carrying the GFP transgene, did not glow green.
Transgenic livestock
• Attempts to use pronuclear microinjection in large animals have had only limited success.
• Development of linker-based sperm-mediated gene transfer (LB-SMGT) has greatly improved efficiency.
Gene pharming
• Turning animals into pharmaceutical bioreactors for protein-based human therapeutics.
• e.g. production of therapeutic proteins in milk or egg white.
15.5 Cloning by nuclear transfer
• The first animal cloning experiments were conducted in the 1950s in the leopard frog, Rana pipiens.
• Briggs and King were interested in directly testing the question of genetic equivalence of somatic cell nuclei.
Genetic equivalence of somatic cell nuclei: frog cloning experiments
• Long-standing question in developmental biology:
– Does cell differentiation depend on changes in gene expression or changes in the content of the genome?
• Nuclear transplantation experiments in Rana pipiens and Xenopus laevis showed that some normal adult frogs could develop from the nuclei of differentiated cells.
• In general, cell differentiation depends on changes in the expression not content of the genome.
Cloning of mammals by nuclear transfer
• A major challenge in performing somatic cell nuclear transfer in mammals is the small size of the mammalian egg.
• Transfers of nuclei from very early embryos to enucleated sheep eggs were not successfully performed until 1986.
• Cloning attempts of nonhuman primates have proved even more difficult.
“Breakthrough of the year:” the cloning of Dolly
• Dolly was the first mammal cloned from an adult cell.
• Less the 1% of all nuclear transfers from adult differentiated cells result in normal-appearing offspring.
The cloning of Dolly confirmed two key principles of genetic equivalence:
1. Differentiated cells on their own are unable to develop into complete animals but the nuclei of most differentiated cells retain all the necessary genetic information.
2. Transfer of a nucleus from a differentiated cells to the environment of the enucleated egg reprograms the nucleus and allows full development.
Method for cloning by nuclear transfer
Four main steps:
1. Preparation of donor cells.
2. Enucleation of unfertilized eggs.
3. Nuclear transfer by cell fusion.
4. Implantation of the embryo into a surrogate mother and analysis of clones.
• DNA typing techniques can be used to confirm that the cloned offspring is genetically identical to the original donor cell nucleus.
Source of mtDNA in clones
• When the cell fusion method is used, the reconstructed embryo will contain egg cytoplasm and the donor nucleus with its accompanying cytoplasm.
• The clone will be heteroplasmic for mtDNA.
Why is cloning by nuclear transfer inefficient?
• To create Dolly, it took 277 trials.
• When 10,000 genes were screened in cloned mice, 4% were shown to be functioning incorrectly.
• Cloned animals suffer from many developmental abnormalities.
• Inefficient reprogramming of the genome.
• Effects of cellular aging.
• Improper segregation of chromosomes during embryonic cell divisions.
Example:
• Rhesus monkey embryos generated by nuclear transfer.
• Missing important components of the mitotic spindle.
Reprogramming the genome
• Totipotent cells are capable of forming any cell type.
• Pluripotent cells are capable of differentiating into several different cell types.
• Differentiated cells are specialized towards a specific function by differential gene expression.
• Tissue-specific genes are activated only in a particular cell type.
• Housekeeping genes are active in most cell types.
• Pluripotency genes are needed for early development but are silent in most adult cell types.
• Successful cloning requires reprogramming of the donor nuclei from differentiated cells to an undifferentiated state.
• Gene silencing is difficult to reverse in cloned embryos (e.g. DNA methylation and imprinting).
Effects of cellular aging
• Dolly the cloned sheep developed arthritis at the relatively young age of 5.5 years.
• Euthanized at age 6 because of complications from a virally induced lung cancer commonly found in older sheep kept indoors.
• Dolly’s cells showed a telomere loss of 20%.
• In contrast to Dolly, telomere length was rebuilt in cloned cattle.
• Telomerase activity was shown to be reprogrammed at the blastocyst stage.
Applications of cloning by nuclear transfer
• Genetically manipulated pets.
• Cloning transgenic animals.
• Cloning of prize animals.
• Wildlife conservation.
• Cloning for stem cells.
Examples of genetically manipulated pets:
• Glofish
• Cloned cats
• Cloned dogs
Cloning of transgenic animals
• Cloned herds of transgenic or gene-targeted farm animals that produce valuable human proteins.
• Cloned herds of agriculturally important animals that are transgenic for a trait of interest.
• Cloning for xenotransplantation.
Cloning of prize animals
Examples:
• Cows with high milk production.
• Champion race horses.
Wildlife conservation
Examples:
• Preservation of endangered species by cloning.
• Trans-species cloning where eggs from the endangered species are not readily available.
Cloning for stem cells
• “Therapeutic cloning.”
• The hope is to develop techniques of growing human ES cells into specific cell types to treat such conditions as Parkinson’s, diabetes, or spinal cord injury.
• A controversial field of research.
• Current research has shown that introducting specific genes or synthetic RNA into adult cells can trigger reprogramming.
• Formation of induced pluripotent stem (iPS) cells.
• The challenge now is to determine how similar or different these iPS cells are compared with human ES cells.
15.6 Transgenic plants
Applications of transgenic technology
• Basic research.
• Increase the performance of commercially important plants by adding new traits or improving on existing ones.
Genetically modified crops: are you eating genetically engineered
tomatoes?
• Genetically modified crops have not always received a warm reception from the public, in part because of human health concerns.
• Example: “Flavr-Savr” tomatoes.
• There are many GM foods in wide distribution, including:– Soybeans– Corn– Canola oil– Cotton seed oil– Hawaiian papayas
• The GM products make up about 80-90% of the market.
Making transgenic dicotyledonous plants is relatively simple procedure for a number of reasons
• Naturally occurring and highly efficient Ti plasmid-based gene delivery system.
• Differentiated plant cells are still totipotent.
• In some species, differentiated plant cells will regenerate into whole adult plants under appropriate conditions.
• Dicotyledons, or dicots, are a class of flowering plants having an embryo with two cotyledons (seed leaves).
– Tomatoes– Potatoes– Beans– Peas– Arabidopsis
• Monocotyledons, or monocots, are a class of flowering plants having an embryo with one cotyledon.
– Daffodils– Lilies– Cereals– Grasses
T-DNA-mediated gene delivery
• Living plants and plant cells in culture can be transformed by transferred DNA (T-DNA) excised from the Ti (tumor-inducing) plasmid.
• Transfer of cloned genes to plant leaf disks is performed using recombinant disarmed Ti plasmids carried by Agrobacterium.
• The leaf disks are transferred to selective shoot- and root-inducing media to form plantlets.
Electroporation and microballistics
• Alternative methods for transfer of cloned genes to plant leaf disks.
• Used for monocotyledonous plants which do not have a natural gene delivery system.
• Electroporation of protoplasts is suitable for some species.
• Microballistic transfection: high-density, subcellular-sized particles are accelerated to high velocity to carry DNA or RNA into living cells.
– Typically gold nanoparticles are fired from a “gene gun.”