This document discusses genetic manipulation techniques for animals, including somatic cell nuclear transfer (SCNT) cloning. It provides details on the SCNT process, including the Roslin technique used to create Dolly the sheep. Applications of SCNT are described for agriculture, conservation, and medical therapeutics. The document also discusses the success of SCNT, limitations, and ethical concerns regarding genetic manipulation of animals.

1. GENETIC MANIPULATION OF
ANIMALS

6. 1. Traditional Techniques
-Pronuclear microinjection
-Through virus
-Isolation and transfusion of primordial germ cells
(PGCs), the embryonic cells that give rise to gametes-
2. Intracytoplasmic sperm injection uses sperm as passive
carriers of recombinant DNA
3. Nuclear Tranfer technology

7. Somatic Cell Cloning

8. Introduction
• Somatic-cell nuclear transfer (SCNT), or Somatic Cell Cloning, is
a laboratory technique for creating a clone embryo with a donor nucleus.
It can be used in embryonic stem cell research, or, potentially,
in regenerative medicine where it is sometimes referred to as
"therapeutic cloning". It can also be used as the first step in the process
of reproductive cloning.
• Because somatic cell cloning is a cloning technique, it is best to outline
principles underlying the process of cloning. Cloning is derived from the
Greek word “klon” which means a twig which can replicate itself and
grows eventually into a tree.

9. • To clone is to reproduce asexually or to
make a copy or a set of copies of an
organism following the fusion or insertion
of a diploid nucleus into an oocyte.
• A true clone is an individual which has all
the components that make up the
individual, including nuclear genetic
material (genome) and other maternally
derived factors that are derived from a
single unique embryo as a result of sexual
reproduction.

10. • In the laboratory, cloning in mammals
involves replacing the genetic material of
an egg with the genetic material of a
somatic cell from an embryo or adult
which will eventually develop into a full
organism or being ; this is a biological
clone; an organism genetically identical
to another organism.

11. • A major advance was made in 1995, when two live
lambs, Megan and Morag (Fig. 13.7), were produced by
nuclear transfer from cultured embryonic cells
(Campbell et al. 1996). This demonstrated the principle
that mammalian nuclear transfer was possible using a
cultured cell line.
History
Fig. 13.7 Megan and Morag, the first sheep
produced by nuclear transfer from cultured
cells. Reproduced by kind permission of the
Roslin Institute, Edinburgh.

12. • The same group later reported the
birth of Dolly (Fig. 13.8), following
nuclear transfer from an adult
mammary epithelial cell line
(Wilmut et al. 1997). This was the
first mammal to be produced by
nuclear transfer from a
differentiated adult cell, and
aroused much debate among both
scientists and the public concerning
the possibility of human cloning
(see Johnson 1998).

13. It was suggested that a critical factor in the success of the
experiment was the quiescent state of the cells in culture, allowing
synchronization between the donor and recipient cell cycles
(reviewed by Wilmut et al. 2002). For the production of Dolly, this
was achieved by lowering the level of serum in the culture
medium, causing the cells to withdraw from the cell cycle due to
lack of growth factors. However, the success rate was very low:
only one of 250 transfer experiments produced a viable lamb, a
phenomenon that has been blamed on a lack of fundamental
understanding of the nuclear reprogramming events that occur
following transplantation (Shi et al. 2003). Similar transfer
experiments have since been carried out in mice, cows, pigs, goats,
cats, dogs, rabbits, mules, and rats using variations on the transfer
methodology developed by Wilmut and colleagues.

14. In the case of Dolly, and possible attempts at
human cloning, the American Medical
Association defines cloning as the production of
genetically identical organisms via somatic cell
nuclear transfer, although a broader definition is
often used to include the production of tissue
and organs from cell or tissue cultures using stem
cells. Somatic cell nuclear transfer refers to the
transfer of the nucleus from an existing organism
into an enucleated oocyte.

15. Somatic cell cloning is not equivalent to fertilsation- In fertilization, the sperm and egg both contain one set of
chromosomes. When the sperm and egg join, the resulting zygote ends up
with two sets - one from the father (sperm) and one from the mother
(egg).
In SCNT, the egg cell's single set of chromosomes is removed. It is
replaced by the nucleus from a somatic cell, which already contains two
complete sets of chromosomes. Therefore, in the resulting embryo, both
sets of chromosomes come from the somatic cell

16. SCNT Process

17. There are two major techniques used in somatic cell
cloning:
The Roslin Technique is a variation
of somatic cell nuclear transfer that
was developed by researchers at
the Roslin Institute. The researchers
used this method to create Dolly. In
this process, somatic cells (with
nuclei in tact) are allowed to grow
and divide and are then deprived of
nutrients to induce the cells into a
suspended or dormant stage. An egg
cell that has had its nucleus removed
is then placed in close proximity to a
somatic cell and both cells are
shocked with an electrical pulse. The
cells fuse and the egg is allow to
develop into an embryo. The embryo
is then implanted into a surrogate.
The Honolulu Technique was
developed by Dr. Teruhiko
Wakayama at the University of
Hawaii.
In this method, the nucleus from
a somatic cell is removed and
injected into an egg that has had
its nucleus removed.
The egg is bathed in a chemical
solution and cultured. The
developing embryo is then
implanted into a surrogate and
allowed to develop

18. In the case of dolly the sheep, the
donor cell was transferred from 10%
fetal calf serum to 0.5% fetal calf serum
for five days, causing it to become
inactive or enter the G0 stage. This
allowed for enucleation and
subsequent implantation of the
somatic cell nucleus into an enucleated
oocyte of a different organism.
The embryo is then implanted
into a surrogate. This step is
thought to mimic the stimulation
normally provided by sperm
during sexual reproduction.

19. A case study: the Life of Dolly
Birth- 5th July
1996
First Wave of Concern:
At one year old, tests
revealed that Dolly's
telomeres were shorter
than those expected for
sheep of that age.
Dolly’s Life: In an attempt to
allow Dolly to have as
normal as life as possible it
was decided that she should
be allowed to breed
6 lambs: Her first
lamb, named Bonnie,
was born in April
1998. The next year
Dolly produced twin
lambs Sally and Rosie,
and she gave birth to
triplets Lucy, Darcy
and Cotton in the year
after that.
Arthritis: In the
autumn of 2001,
at the height of
the Foot and
Mouth outbreak
in the UK, Dolly
was seen to be
walking stiffly
Dolly’s Final Illness:
In January 2000, one
of the cloned sheep,
Cedric, died. The post
mortem revealed
that Cedric had died
of sheep pulmonary
adenomatosis (SPA).
Death: 14th
of February
2003

20. Applications of Somatic Cell Cloning
• A key reason behind the usefulness of cloning
is that by producing near-identical genetic
copies of an organism, results are faster and
more predictable than in previous
reproductive techniques like artificial
insemination, which involve costly and
potentially harmful procedures such as
cryopreservation.
• Many of these procedures require the use of
stem cells.

21. Agriculture:
• SCNT ensures the rapid production
of genetically modified herds or elite
individuals with desirable traits,
eg. For milk containing extra
nutrients or meat more consistent
in taste and quality.
• It also allows genetic conservation of
local breeds with unique tolerance
for regional diseases or local
climates. This approach was used
to clone the last surviving Enderby
Island cow from mural granulosa
cells.
• SCNT also allows spread of disease
resistance faster than traditional
techniques. For instance, herds of
clones lacking the prion protein
gene will no longer be susceptible
to bovine spongiform encephaliti, also
known as the Mad Cow disease.
Conservation:
• Conservation has been
highlighted recently as an area
where the SCNT technique may
be useful. It may preserve and
propagate endangered species that
reproduce poorly in zoos until
their habitats can be restored and
populations reintroduced to the
wild. Attempts have been made
with the Giant Panda for instance.
The technique allows maintenance
or increase of the overall genetic
diversity of a species by introducing
new genes from preserved
specimens or animals in other
wild and captive populations of
the same species back into a
diminishing gene pool.
SCNT may even recreate
extinct species, if viable
tissues/cells have been
banked or are available.
An example is the
mammoth, where an intact
animal was discovered
frozen in the Tundra
recently; the closely related
elephant can be used as
both oocyte donor as well
as surrogate mother.

22. Medical Therapeutics:
• The greatest potential of the SCNT technique is in medical
therapeutics and this is therapeutic cloning. The source cell can
be human or animal, though the patient’s own cells will be the
most likely source for therapeutic cloning.
Therapeutic cloning can be categorized into:
• a. Replacement tissues & organs;
• b. Prevention of immunological tissue rejection, to allow for
more successful organ transplantation;
• c. Enhancement of immunological surveillance, to prevent
cancers; and
• d. Gene therapy, to correct genetic defects by introducing the
functional gene (probably through stem cell re-population)
• Clinical Applications include the ability to prevent, treat and
overcome: Aging, Disease, Cancers, Myocardial infractions and
Genetic disorders amongst many others.

23. Clinical Applications can be grouped into two
categories:
• Fine-tuning of existing strategies, eg.
production of and screening for new drugs;
and new strategies, also known as “cell-based
therapy”
• Use is in a rapidly growing field of medicine,
known as regeneration medicine. “Pharming”
the production of pharmaceuticals by
extracting and purifying desired molecules
from the milk of genetically modified
livestock is an example of the production of
new drugs and proteins

25. The Success of SCNT procedures
• Cloning ensures the presence of a trans-gene by
introducing the DNA into the somatic cell lines in
culture instead of by the more traditional and
tedious process of altering individual genotypes.
Cattle producing insulin in their milk are an
example.
• The first example published was Polly, another
lamb cloned by the Roslin Institute. She was
derived from fetal skin cells, genetically modified
to contain a human gene. This has resulted in a
valuable sheep that secretes human factor IX in its
milk. The blood-clotting protein is extracted,
purified, and used to treat haemophilia B.
SCNT is being used to produce human proteins for
therapy. Human proteins are in great demand for the
treatment of a variety of diseases. Whereas some can
be purified from blood, this is expensive and runs the
risk of contamination by HIV or Hepatitis C. Proteins
can be produced in human cell culture but costs are
very high and output small. Much larger quantities
can be produced in bacteria or yeast but the proteins
produced can be difficult to purify and they lack the
appropriate posttranslational modifications that are
needed for efficacy in vivo.
-SCNT can be used in cancer treatment by cloning
cells from cancerous tissue and introducing specific
characteristics leading to early cell death (eg. Short
telomeres). Reintroducing the altered cells could
decrease the capacity for division and replication in
the tumour.
- SCNT can also be useful in biomedical research.
Large animals can be genetically modified to carry
genetic defects mimicking human illnesses such as
cystic fibrosis. The similarities in organ size and life
span allow for improved monitoring of factors such as
the long-term consequences of treatment.
SCNT can also be used in xenotransplantation ( Transfer of living
cells, from one species to another) The chronic shortage of organs
means that only a fraction of patients who could benefit actually
receive transplants. Genetically modified pigs are being developed
as an alternative source of organs by a number of companies,
though so far the modifications have been limited to adding genes.
Nuclear transfer will allow genes to be deleted from pigs and much
attention is being directed to eliminating the alpha -galactosyl
transferase gene. These encode an enzyme that creates
carbohydrate groups which are attached to pig tissues and which
would be largely responsible for the immediate rejection of an
organ from a normal pig by a human patient.

26. Limitations
• A limitation of the SCNT technique for human therapeutic cloning
is the need for human oocytes. Hence, the “universal donor” oocyte,
Animal oocytes have been postulated for use with human somatic
cells to create hybrid embryos, but this approach has not been
accepted by many.
• Another limitation is the need for feeder cells to maintain the stem
cells in an undifferentiated state.
• In SCNT, not all of the donor cell's genetic information is
transferred, as the donor cell's mitochondria that contain their
own mitochondrial DNA are left behind. The resulting hybrid cells
retain those mitochondrial structures which originally belonged to
the egg. As a consequence, clones such as Dolly that are born from
SCNT are not perfect copies of the donor of the nucleus
• Stresses placed on both the egg cell and the introduced
nucleuses are enormous, leading to a high loss in
resulting cells. For example, Dolly the sheep was born
after 277 eggs were used for SCNT, which created 29
viable embryos.
• Only three of these embryos survived until birth, and
only one survived to adulthood. As the procedure
currently cannot be automated, but has to be performed
manually under a microscope, SCNT is very resource
intensive. The biochemistry involved in reprogramming
the differentiated somatic cell nucleus and activating
the recipient egg is also far from understood

27. Ethical Concerns
SCNT AS UNETHICAL
• many believe that the use of somatic cell
nuclear transfer in reproductive cloning
defies the natural way of conceiving
children.
• Reproductive SCNT would devalue the
genetic distinctiveness of each
individual. It would deprive the child of
a sense of mystery or right to ignorance
about his or her origin
• Religious groups do also believe that
the use of technology to create a human
clone is ‘playing with God’ and
therefore is religiously, and morally,
unacceptable
SCNT AS ETHICAL
• Somatic cell nuclear transfer
procedures have been posed as ethical
in 2 situations:
• Infertile couples who cannot otherwise
be treated
• Couples at risk of passing a serious
genetic disease on to their children.
If both the male and female partners
are carriers of autosomal-recessive
disease traits, one partner’s somatic
cell could be used to conceive. If one
partner has an autosomal- dominant
disease trait, the unaffected partner
could provide the somatic cell.