Introduction to Gene Cloning

1. UNIT 4:
What is Cloning?
Clones are organisms that are exact genetic copies. Every single bit of their
DNA is identical.
Clones can happen naturally—identical twins are just one of many examples. Or
they can be made in the lab. Below, find out how natural identical twins are
similar to and different from clones made through modern cloning technologies.
How Is Cloning Done?
Many people first heard of cloning when Dolly the Sheep showed up on the
scene in 1997. Artificial cloning technologies have been around for much longer
than Dolly, though.
There are two ways to make an exact genetic copyof an organism in a lab:
artificial embryo twinning and somatic cell nuclear transfer.
1. Artificial Embryo Twinning
Artificial embryo twinning is a relatively low-tech way to make clones. As the
name suggests, this technique mimics the natural process that creates identical
twins.
In nature, twins form very early in development when the embryo splits in two.
Twinning happens in the first days after egg and sperm join, while the embryo
is made of just a small number of unspecialized cells. Each half of the embryo
continues dividing on its own, ultimately developing into separate, complete
individuals. Since they developed from the same fertilized egg, the resulting
individuals are genetically identical.
Artificial embryo twinning uses the same approach, but it is carried out in a
Petri dish instead of inside the mother. A very early embryo is separated into
individual cells, which are allowed to divide and develop for a short time in the
Petri dish. The embryos are then placed into a surrogate mother, where they
finish developing. Again, since all the embryos came from the same fertilized
egg, they are genetically identical.

2. 2. Somatic CellNuclearTransfer
Somatic cell nuclear transfer (SCNT), also called nuclear transfer, uses a
different approachthan artificial embryo twinning, but it produces the same
result: an exact genetic copy, or clone, of an individual. This was the method
used to create Dolly the Sheep.
What does SCNT mean?
Somatic cell: A somatic cell is any cell in the bodyother than sperm and egg,
the two types of reproductive cells. Reproductive cells are also called germ
cells. In mammals, every somatic cell has two complete sets of chromosomes,
whereas the germ cells have only one complete set.
Nuclear:The nucleus is a compartment that holds the cell's DNA. The DNA is
divided into packages called chromosomes, and it contains all the information
needed to form an organism. It's small differences in our DNA that make each
of us unique.
Transfer:Moving an object from one place to another. To make Dolly,
researchers isolated a somatic cell from an adult female sheep. Next they
removed the nucleus and its entire DNA from an egg cell. Then
they transferred the nucleus from the somatic cell to the egg cell. After a
couple of chemical tweaks, the egg cell, with its new nucleus, was behaving just
like a freshly fertilized egg. It developed into an embryo, which was implanted
into a surrogate mother and carried to term. (The transfer step is most often
done using an electrical current to fuse the membranes of the egg and the
somatic cell.)
The lamb, Dolly, was an exact genetic replica of the adult female sheep that
donated the somatic cell. She was the first-ever mammal to be cloned from an
adult somatic cell.
How does SCNT differ from the natural way of making an
embryo?
Natural fertilization, where egg and sperm join, and SCNT both make the same
thing: a dividing ball of cells, called an embryo. So what exactly is the
difference between the two?
An embryo's cells all have two complete sets of chromosomes. Thedifference
between fertilization and SCNT lies in where those two sets come from.

3. In fertilization, the sperm and egg have one set of chromosomes each. When the
sperm and egg join, they grow into an embryo with two sets—onefrom the
father's sperm and one from the mother's 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. So, in the resulting embryo, both sets of chromosomes come
from the somatic cell.
Is cloning an organism the same as cloning a gene?
You may have heard about researchers cloning, or identifying, genes that are
responsible for various medical conditions or traits. What's the difference?
When scientists clone an organism, they are making an exact genetic copy of
the whole organism, as described above.
When scientists clone a gene, they isolate and make exact copies of just one of
an organism's genes. Cloning a gene usually involves copying the DNA
sequence of that gene into a smaller, more easily manipulated piece of DNA,
such as a plasmid. This process makes it easier to study the function of the
individual gene in the laboratory.

4. Reproductive Vs Therapeutic Cloning
What is therapeutic cloning?
Therapeutic cloning refers to the removal of a nucleus, which contains the
genetic material, from virtually any cell of the body (a somatic cell) and its
transfer by injection into an unfertilised egg from which the nucleus has also
been removed. The newly reconstituted entity then starts dividing. After 4-5
days in culture, embryonic stem cells can then be removed and used to create
many embryonic stem cells in culture. These embryonic stem cell ‘lines’ are
genetically identical to the cell from which the DNA was originally removed.
Therapeutic cloning is also known as somatic cell nuclear transfer (SNCT) as
the term cloning is frequently misunderstood by the general public. The word
‘cloning’ more often conjures up thoughts and beliefs about reproductive
cloning. Reproductive cloning is if a newly formed embryo, resulting from a
therapeutic cloning procedure, were transferred into the womb of a woman, it
could theoretically, develop into a fetus. This technique has been used to clone
agricultural animals, endangered species and recently domestic pets and
primates, but has not been proven in humans. The scientific community
overwhelmingly rejects the use of therapeutic cloning for the purposes of
human reproductive cloning. Reproductive cloning is illegal in Australia and in
many other countries. While the procedure of therapeutic cloning employs
aspects of cloning technology, researchers today are interested in therapeutic
cloning as a means of deriving human embryonic stem cell lines for use in
research and, ultimately, therapy. Therapeutic cloning has been legal in
Australia since 2006 under the Prohibition of Human Cloning for Reproduction
and the Regulation of Human Embryo Research Amendment Act 2006. Any
Australian researchers wishing to use therapeutic cloning must apply to the
National Health & Medical Research Council (NHMRC) for a licence.
What is special about therapeutic cloning?
The capacity of therapeutic cloning to re-program adult nuclei is extraordinary
and unique. Cells of particular tissues generally express a characteristic set of

5. genes. Whether they are primitive stem cells, fully-differentiated (i.e. mature)
cells, or something in between these extremes. Particularly for more mature
cells, the tissue-specific patterns of gene expression are quite stable through
many rounds of cell division. Upon transfer to an enucleated egg, the adult
nucleus becomes re-programmed in the environment of the egg. That is, genes
that were not used before (switched off) become reactivated. A poorly-
understood process, re-programming involves dramatic changes in the pattern of
genes which are active in the nucleus. Instead of the adult nucleus causing the
egg to behave like an adult cell, the egg causes the nucleus to go backwards
along a differentiation sequence, resulting in an embryonic type cell. As a result
of therapeutic cloning, the previously unfertilised egg takes on the properties of
a fertilised egg and begins the first stages of development into an embryo.
What is known about the embryos resulting from therapeutic
cloning?
In broad terms, embryos arising from therapeutic cloning are the same as
embryos from fertilisation of an egg by a sperm. In agricultural research,
therapeutic cloning has been used to create embryos in the laboratory which
have been transferred into animals and given rise to offspring. 'Dolly' the sheep
was born as a result of this procedure. However, there are also differences
between a normally fertilised egg and one produced by therapeutic cloning,
which are not generally understood. In animal studies clones appear to have
increased abnormality and decreased pregnancy rates. For example, despite
being relatively young for a sheep, which can live to 11 or 12 years of age,
Dolly died prematurely at the age of six years, showing signs of arthritis and
lung infection. Other than the major ethical concerns, this is a fundamental
reason why reproductive cloning should not be performed on humans. Research
involving therapeutic cloning and its potential application .While the scientific
community overwhelmingly rejects the use of THERAPEUTIC CLONING for
reproductive cloning, it would provide an invaluable tool for basic research. As
reported in Nature in November 2007 scientists successfully extracted stem
cells from nonhuman cloned primate embryos. Although it is a highly
significant achievement it must be considered that it took 304 eggs to produce
two successful embryonic stem cell lines. In January 2008 a Californian
company Stemagen reported it had successfully cloned a human blastocyst, an
early stage embryo. The Stemagen embryos were the first to be made with
human adult skin cells through therapeutic cloning, however they did not

6. attempt to produce stem cell lines, focusing their attention on extensive genetic
tests to prove the identity of the cloned embryos. Researchers regard therapeutic
cloning as an effective method for deriving human embryonic stem cells with
specific characteristics, about which a great deal remains to be known and
understood. The promise of therapeutic cloning is that it will be an effective
way to derive embryonic stem cells which can then be used for the development
of patient- and diseasespecific cell-based therapies as well as the production of
stem cells with specific disease characteristics for research purposes. The use of
a patient's own cells for tissue replacement through therapeutic cloning
overcomes the problem of immune rejection that is a major complication of
tissue transplantation today. Embryonic stem cells derived by nuclear transfer
may, in the future, be used to treat diseases including diabetes, heart disease and
Parkinson’s disease.
Reproductive Cloning
This type of cloning is done with the intent of making another organism. The
clone will be an exact duplicate of the one that exists now. Reproductive
cloning includes animals, plants, and humans.
Somatic Cell Nuclear Transfer, also referred to as SCNT, is the technique used
for this type of cloning. A recipient egg's genetic material is extracted so that
only an empty egg remains. A cell from the organism being cloned is taken and
its nucleus is extracted. This nucleus is then implanted in the egg. The egg is
then forced to divide, using a mild electric shock or chemicals, creating an
embryo which will eventually be placed into the uterus of a surrogate mother.
As of today, this type of cloning has been used for research only, but its future
uses are staggering. This type of cloning could be beneficial in making breeding
specific animals simpler or in repopulating endangered species. It may also be
used to create organisms with specific characteristics like genetically unique
animals or drug-producing animals.
Ethical Issues of Cloning
Ever since the advent of cloning, there have been arguments for and against this
process. A fundamental argument is that cloning is ethically wrong and various
religious groups have rejected it saying that cloning is equivalent to 'playing
God'. Here is a discussionabout the ethical issues that have arisen concerned
with cloning humans.

7. The rapid advancements in science and technology over the last couple of
decades has meant that mankind is exploring newer frontiers and challenging
long-held beliefs and notions. One such field is cloning. Creating exact copies
or cloning human beings has always fired the human imagination. This desire
has manifested itself in various art and entertainment depicting cloned humans.
The successfulcloning of Dolly in 1997 further fueled talk about the possibility
of human cloning. Over the years, cloning has come to mean an artificial and
identical genetic copyof an existing life form.
To explain in a scientific way, cloning means replacing the egg nucleus of an
organism with the donor's nucleus. This nucleus contains unique genes of the
donor. The procedureinvolves removing the nucleus of a somatic cell and
inserting it into an enucleated or unfertilized egg cell. Unlike natural
reproduction, wherein the egg contains a combination of genetic material, this
egg which grows into an embryo contains only the donor's gene.
Theoretically, this might seem fairly straightforward. However, a high failure
rate along with prevalence of high deformity and disability rates in cloned
animals, strongly suggests cloning might not be applicable to humans.
The Ethical Issues
Religious Beliefand Control:
Cloning goes against the basic belief of certain religions that only God has
created life and its various forms in nature. Humans cannot act as God. Even
when genetically identical twins are born, their embryo splits spontaneously or
randomly to give a new unique genetic combination. Cloning involves a
controlled split of the embryo to produce a tailor-made genetic makeup.
Ethically, it is wrong for any human to have control over the genetic makeup of
any other individual. More so, the cloned individual would be generated for
specific purposes. This in essence is wrong wherein the purpose of an
individual's life should be more than just satisfying someone else's needs.

8. Relationships and Individuality
Cloning creates a new human, yet strips him off his individuality. A man, along
with his clone can never be dignified as a single identity. The uniqueness
attributed to humans from God might be at stake. The replication of an
individual is a major blow to his most distinct feature - his identity. Another fact
is that we are unsure how the cloned individual might react and behave with
regards to his family and parents. Furthermore, if the cloned individual is cloned
from his grandparents and not his parents, would he/she be considered a
sibling? How would he/she react? How would the parents and family regard the
cloned individual? When we are unsure about the implications or consequences
of such situations, it is ethically wrong to subject any individual to suchtests as
fellow human beings.
Failure Rate
Physicians and doctors have a moral obligation to ensure and translate the safety
of any medical procedureto his/her patients. As of now, no one can guarantee
that the child born due to cloning would be a healthy one. As indicated earlier,
the high failure rate in cloning mammals and other species is completely
unacceptable when it comes to cloning humans. Moreover, in case of a failed
cloning attempt, putting down mammals or other species in itself is distressing.
Translating the same in case of human clones is ethically and medically
unjustifiable, as well as criminal.
Legal and Other Issues
Besides the above mentioned issues, there are other issues which may seem
technical as of now, but can arise out of lack of knowledge or unforeseeable
circumstances.
Altering Gene Pool
If cloning becomes widespread, the genetic diversity of humans will go down.
This would result in the decrease in immunity of humans against diseases. Thus
making humans susceptible to epidemics and unknown diseases. Some advocate
human cloning as ethically unacceptable becauseit is seen as a threat to the

9. entire human evolution. Though this issue is slightly hypothetical, it still can
posea potential threat to all humanity. Along with reducing generic diversity,
there are risks of transmitting degenerative diseases from the donorhuman to
the clone. Trans-genetic manipulation, where genetic material from one species
is artificially inserted into another species, if applied to humans, would lead to
transfer of diseases from other species. Thus, large-scale cloning might prove to
be a serious blow to the entire human race in future.
Illegal Cloning and Clones
Cloning could have legal implications as well. A cloned child having multiple
donors might complicate parental right issues as well as inheritance and marital
eligibility issues. Another view held by many experts, suggests that there is a
possibility of clones being developed without the concerned individual's
consent. This will definitely create legal issues not to mention violation of
medical as well as moral ethics. Many people are also concerned that clones
would be produced with a specific need and purposein mind and such cloned
individuals would be traded or sold, amounting to human trafficking which is
illegal.
At the other end of spectrum are some experts who are of the opinion that the
embryo does not require any particular moral consideration. They say that, at
the stage when an embryo is cloned, it is just a bunch of cells that contain DNA,
which are not very different from the millions of skin cells that we shed every
day. The embryonic cells at that stage cannot be considered equivalent to a
human being becauseit does not have thoughts, self-awareness, memory,
awareness of its environment, sensoryorgans, internal organs, legs, arms, and
so on. They think that the embryo attains human identity or individuality much
later during gestation, perhaps at the point when the brain develops so that it
becomes aware of itself.
In view of the highly debatable aspects aboutcloning and weighing in on the
pros and cons of this process, UNESCO passed a non-binding United Nations
Declaration on Human Cloning, in March 2005, which states: Practices which
are contrary to human dignity, such as reproductive cloning of human beings,
shall not be permitted. In the United States there are no federal laws that ban
cloning completely, yet 13 states have banned reproductive cloning. Although
many countries have banned cloning, many countries allow therapeutic cloning,

10. a system in which the stem cells are extracted from the pre-embryo, with the
intention of generating a whole organ or tissue, so that it can be transplanted
back into the person who gave the DNA.
Since human cloning raises some serious concerns, it would be highly
irresponsible to pursue this method, without giving it a serious thought. New
issues are bound to crop up with advances in this field, and only time can decide
its fate. Until the benefits are discussed by society to outweigh the harm, it
would be inappropriate to participate in cloning of humans.