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Animal Biotechnology
Dr. Sabina Semiz, PhD
Assoc. Professor
31. 03. 2009.
IUS 2008-2009
Biotechnology
(BIO 401)
4SS BIO401
Animal Biotechnology
Introduction to Biotechnology (Pearson 2nd Edition)
Chapter 7, p. 171.
Learning Objectives
List some of the medical advances made using animal research models.
Explain what makes a good animal model for genetic studies.
Describe two alternatives to the use of animal models, including their limitations.
Discuss some of the ethical concerns about using animals in research.
Outline the process used to create Dolly the sheep.
Discuss some of the limitations to the cloning processes.
List some of the products that can be produced using transgenic animals as bioreactors.
Explain how knockouts animals can be used to provide information about genetic disorders and other diseases.
What is a Clone?
A cell or collection of cells that is genetically identical to another cell or collection of cells.
Animals that reproduce asexually are clones of a parent.
Identical twins are clones of each other.
"It is not a simple life to be a single cell, although I have no right to say so, having been a single cell so long ago myself that I have no memory at all of that stage of my life."
—Lewis Thomas (1913–1993) author, biologist, physician

Dolly
Dolly with her first newborn, Bonnie
Born in July 1996 at the Roslin Institute in Scotland.
First mammal to be cloned from an adult mammal using the nuclear transfer technique
277 attempts were made before the experiment was successful
Dolly died in February 14, 2003 of progressive lung disease at the age of 6; whereas normal sheep can live up to 12 years of age.
Dolly with her surrogate mother
http://www.faseb.org/opar/cloning/cloning.htm
Nuclear Transfer to Produce
Cloned Animal
Cloning usually begins with removal of the egg nucleus from a fertilized egg and replacement with another nucleus (possibly from an adult animal like a cow).
Mammal Cloning Timeline
http://www.cnn.com/2001/WORLD/europe/08/06/clone.critics/index.html
Megan and Morag
Dolly
http://hs.houstonisd.org/hspva/academic/Science/Thinkquest/gail/text/benefits.html
ANDi, the first transgenic rhesus monkey, carries the gene for green fluorescent protein but does not glow green.
Safety of Cloned Animals
In January 2008 the FDA approved the food produced from cloned cattle, goats, and pigs as safe.
These animal products can be sold in markets and restaurants without specila labels.
The market for cloned animals in the US is expected to reach nearly $50 mill. annually.
Mammal Cloning allows propagation of endangered species
January 8, 2001 Noah, a baby bull gaur, became the first clone of an endangered animal.
Comparison of Cloning Success Rates in Various Animals
The table shows success rates of cloning when mature mammal cells were used.
Yanagimachi, R.  2002.  "Cloning: experience from the mouse and other animals." Molecular and Cellular Endocrinology.  21 March, 187.
Clone Birth Defects
Cloned offspring often suffer from large offspring syndrome, where the clone and the placenta that nourished it are unusually large.
Cloned offspring often have serious inexplicable respiratory or circulatory problems, which causes them to die soon after birth.
Clones tend to have weakened immune systems and sometimes suffer from total immune system failure.
Very few clones actually survive to adulthood.
Clones appear to age faster than normal.
Clones experience problems associated with old age, such as arthritis, while they are still young.
This may be due to the fact that clones have shorter telomeres
The whole story about cloning is not a reproductive story
The possibility of using cloning technology
to grow organs genetically identical
to our own for transplantation –
thereby avoiding rejection of foreign issues
http://medlib.med.utah.edu/WebPath/CVHTML/CV001.html
Greatest danger:
Clones being harvested for their body parts
Knock-out animals and
Transgenic animals
ES cells
Transgenic Animals:
Animal biotechnology is the field to engineer transgenic animals, i.e., animals that carry genes from other species.
The technology has already produced transgenic animals such as mice, rats, rabbits, pigs, sheep, and cows.
What is a transgenic animal?
A transgenic animal is one whose genome has been changed to carry genes from other species.
For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene introduced which can knock out the functioning of another particular gene in the embryo.
Transgenic Animals
Animals that have their DNA manipulated in this way are known as transgenic animals.
Transgenic animals are useful as disease models and producers of substances for human welfare.
Why are these animals being produced?
Some transgenic animals are produced for specific economic traits.
E.g., transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema.
Transgenic Animals
Other transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied).
The OncoMouse® or the Harvard mouse, carrying a gene that promotes the development of various human cancers.
Human Gene Targets Begin
with Animal Gene Discovery
and Testing
How are transgenic animals produced?
DNA microinjection
Introducing the transgene DNA directly into the zygote at an early stage of development.
No vector required.

Retrovirus-mediated gene transfer:
Infecting mouse embryo with a retrovirus which carry the new gene.
Using virus as a vector .
Embryonic stem cell-mediated gene transfer
The blastocyst (inner layer of a fertilized egg) is harvested and mixed with recombinant DNA and inserted back in the blastocyst.

Sperm-mediated transfer
Use of “Linker protein" to attach DNA to sperm which transfer the new DNA during fertilization.
Gene gun
Embryonic stem cell-mediated gene transfer
This method involves:
Isolation of totipotent stem cells (stem cells that can develop into any type of specialized cell) from embryos.
The desired gene is inserted into these cells.
Cells containing the desired DNA are incorporated into the host`s embryo.
Zebrafish – Common Animal Model
Transplanting genes into their embryos (a) allows study of the developmental changes (Like blood vessel growth) before they become adults.
Transgenic Animals
Definition:  An organism (typically a mouse) that is engineered to carry a foreign gene, or transgene of choicem as part of its own genetic material.
Purpose:  These animals are very useful for delineating the function of newly discovered genes as well as for producing useful proteins in large animals.
In some of the eggs, the genetic material integrates at a random site on a chromosome and so becomes part of the mouse cell`s genetic material the animal resulting from that egg will therefore carry that gene and so is referred to as a "transgenic animal".
Procedure for Producing Transgenic Mice


Three different breeding pairs of mice are required.
First Breeding Pair:
Fertile male + superovulated female
Fertile male
Superovulated female = immature female induced to superovulate
Pregnant mare’s serum (=FSH) on day 1
Human Chorionic Gonadotropin (=LH) on day 3
Mated on day 3
Fertilized oocytes microinjected on day 4 with foreign DNA construct.
Microinjected oocytes are transferred to the oviducts of surrogate mothers at end of day 4.

Procedure for Producing Transgenic Mice
Second breeding pair:
Sterile male + surrogate mother
Sterile male produced through vasectomy
Surrogate mother must mate to be suitable recipient of injected eggs
Mated on day 3
Microinjected oocytes from first breeding pair are transferred to oviducts on day 4
Embryos implant in uterine wall and are born 19 days later.
Southern blotting techniques confirm presence and copy number of transgenes.
Procedure for Producing Transgenic Mice
Third breeding pair:
Foster parents
Fertile male + female mated to give birth on same day surrogate mother
Serves as foster parent if caesarian section is required for surrogate mother

Procedure for Producing Transgenic Mice
Embryonic stem (ES) cells
Pluripotent stem cells derived from the inner cell mass of the blastocyst
Can be cultured, manipulated and then reinjected into blastocysts,
where they can go on to contribute to all parts of embryo.
In principle, ES cells
also
might be able
to generate
large quantities
of any desired cell
for transplantation
into patients.
Totipotent and
pluripotent cells
Totipotent =
meaning that
its potential is total.
pluripotent =
they can give rise
to many types of cells
but not all types of cells
(no fetus developed).
isolated directly
from the inner cell mass
of embryos
at the blastocyst stage.
(IVF-IT surplus embryos
in case of humans)
More about stem cells
Embryonic stem cells
Adult stem cells
Truly pluripotential
More restricted
pattern of differentiation
medical gain without ethical pain
several countries
have sanctioned deriving
human ES-cell lines
from ‘surplus’ embryos
created through
in vitro fertilization
although several human
ES-cell lines have been made,
they will not be immunologically compatible with most patients
who require cell transplants.
Transgenic mice
The growth hormone gene has been engineered to be expressed
at high levels in animals.
The result: BIG ANIMALS
metallothionein promoter
regulated as heavy metals
Mice fed heavy metals are 2-3 times larger
Studies Utilizing Transgenic Mice
“Pharm” animals
(transgenic livestock)

Bioreactors whose cells have been engineered to synthesize marketable proteins

DNA constructs contain desired gene and appropriate regulatory sequences (tissue-specific promoters)

More economical than producing desired proteins in cell culture

Antifreeze gene promoter
with GH transgene in atlantic salmon
GH gene comes from
larger salmon
Wild and domestic trout respond differently
to overproduction of growth hormone.
So in some cases, GH not effective.
Problem with GH fish
Transgenic salmon will escape from fisheries
and breed with strains in the wild ?

If the transgenic fish have
a mating advantage (not clear)
and are less fit (which they are),
their offsprings will produce negative effect
on the normal population.
Solutions:

1) To grow sterile fish
2) To grow fish inland without chances to escape in the wild
Improving Agricultural Products with Transgenics
Transgenic technology holds great potential in agriculture, medicine, and industry
The benefits of these animals to human welfare can be grouped into areas:
Agriculture
Medicine
Industry
1. Agricultural Applications
Breeding
Traditional cross breeding have been used for ages to create chickens, cows, pigs etc.
Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate).
Traditional breeding is a time-consuming, difficult task.
Researchers have now used gene transfer to improve the productivity of livestock.
Now it is possible to develop traits in animals in a shorter time and with more precision.
It also offers farmers an easy way to increase yields.

Scientists can improve the size of livestock genetically.
Transgenic cows exist that produce more milk or milk with less lactose or cholesterol.
Transgenic cows have been used to produce milk which are richer in proteins and lower in fat.
B) Quality
Herman, a transgenic bull carries a human gene for Lactoferrin (gene responsible for higher iron content)
Pigs and cattle that have more meat on them.
Sheep that grow more wool.
Eggs can be made healthier with high quality protein.
C) Disease resistance
Disease-resistant livestock is not a reality just yet.
But there has been improvement in disease reduction in animals.
The Foot- and- Mouth disease in England in 2000 led to destruction of herds of cattle, sheep and goat.

Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.
Transgenic disease protection promises a long term cost effective method of battling animal diseases.
2. Medical Applications
Xenotransplantation

Transplant organs may soon come from transgenic animals.
B) Nutritional supplements and pharmaceuticals

Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats.
The first transgenic cow (Rosie ) produced human protein-enriched milk at 2.4 grams per liter.


This transgenic milk is a more nutritionally balanced product than natural milk and could be given to babies or the elderly with special nutritional or digestive needs.
A transgenic cow exists that produces a substance to help human red cells grow.
C) Human gene therapy

Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene.
Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.
3. Industrial Applications
:

By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.
Biosteel is an extraordinary new product that may be soon used in bullet proof vests and in suture silk for stitching wounds.
Animals have been used as “Bioreactors” to produce proteins. Genes for desired proteins are introduced via transgenics to the target cells .
:

The target cells are cloned and several such cells are raised into adults.
These adults may produce milk or eggs (due to the presence of introduced gene rich in desired protein).
Toxicity-sensitive transgenic animals have been produced for chemical safety testing.
Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.

Transgenic animals have been used to produce pharmaceutical protein: example a human gene called AT III has been transferred to goats.
Goats milk contain this protein that prevents blood clotting (goats multiply faster than cows)
“Hen bioreactor” eggs are used to enrich protein by recombinant DNA technology.
Transgenic Goats That Produce Valuable Proteins in Their Milk – “Biorectors”
Transgenic Models in Drug Development
Search for new drug targets
Validation of drug targets
Safety testing
Protein production
Unexpected Phenotypes
Phenotype more severe than expected:
Early lethal
Lack of inductive signals
Phenotype less severe than expected:
Incomplete gene disruption
Genetic redundancy
Functional redundancy (compensation)
Models for Target Validation

Disease Models
Reporter mice to monitor gene expression
Mice with human drug targets (eg. knock-in mice)
What are the ethical concerns surrounding transgenesis?
Ethical concern is ever increasing as the technology grows, including the issue of lab animal welfare
These ethical issues include questions such as:
Should there be universal protocols for transgenesis?
Should such protocols demand that only the most promising research be permitted?
Is human welfare the only consideration?
What about the welfare of other life forms?
Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering?
A Summary of Animal Cloning


Although there has been limited success in cloning some animals, it`s still seen as a viable technology.
Ever since the announcement of the birth of Dolly, additional sheep, cows, goats, pigs, and mice have been cloned.
:

There are still obvious problems as evidenced from the numerous deaths of cloned animals that occur just before or after birth.
Cloning is a big first step. Genetic manipulation of cloned animals is the future direction of the cloning frontier.

Cloning can produce genetically identical laboratory animals which can be used as models for human disease.
No live dog clones have yet been reported, the company PerPETuate, Inc. (Connecticut) is freezing tissue from family pets for the future.
Cloned Kittens
4-month-old cloned cats in 2004 (CA, US).
:

The cloning of human embryos for reproductive purposes is illegal at this time.
It is still important to examine the consequences and the likelihood of this scenario.
Identical twins illustrate that being genetically identical does not remove their humanness.

At any rate, there have been significant difficulties with cloning primates, including an extremely low success rate and a high number of abnormalities.
These results make it unacceptable to attempt human cloning at this moment in time.
Cloning has opened many doors that could lead to remarkable medical advancements but, as with all new technologies, it will be accompanied by ethical and social dilemmas.