Download Genetically Modified Organisms: Use in Basic and Applied

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 tissuespecific 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 genetargeting 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 normalappearing 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.”