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.”