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.”