11111 Fact Sheet 22 CLONING AND STEM CELLS 1 1 .docx

11111 Fact Sheet 22 | CLONING AND STEM CELLS
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www.genetics.edu.au Updated 30 September 2015
This fact sheet describes the process of cloning and producing
stem cells. It outlines the potential
benefits, ethical challenges, and provides an overview of the
current policies governing the technology.
In summary
not yet differentiated into a
specific tissue or organ cell
segment of DNA,
a cell or a whole organism
of an existing organism
e.g. Dolly the sheep
cells that can then be
turned into a specific cell type needed to repair or replace
damaged tissue

Therapeutic cloning is allowed
only for medical research that is governed and overseen by the
National Health and
Medical Research Committee (NHMRC).
WHAT IS A STEM CELL?
Stem cells are cells that have several important
properties that allow them to repair or replace
damaged tissue or organs:
division
able to develop into different cell types for
different tissues and organs.
WHAT IS CLONING?
Cloning is the creation of an exact genetic replica
of a small segment of DNA, a cell or a whole
organism.
clones that are created naturally
created artificially in a laboratory in Scotland
in 1997.
Dolly’s creation led to international awareness of
cloning but there are several different types of
cloning that can be used for purposes other than
producing a genetic replica (or identical twin) of a
whole organism like humans or sheep.

TYPES OF CLONING
(a) DNA Cloning
DNA cloning is also known as recombinant DNA
technology, molecular cloning and gene cloning.
It is used to produce many copies of a particular
segment of DNA containing one or more genes so
that the activity or function of the DNA can be
studied in the laboratory.
How is DNA cloning done?
The DNA segment of interest from an organism
such as a human is incorporated into the plasmid
DNA of a bacterial cell. A plasmid is a circular self-
replicating DNA molecule that is separate from the
bacterial DNA (Figure 22.1).
The plasmid containing the genes or DNA of
interest is now a piece of recombinant DNA, made
up of human and bacterial DNA. It is then put into
a cell that will act as a host: as the host cell is
copied over and over again, the recombinant DNA
is copied as well. Bacteria are most often the host
cells but yeast and mammal cells can be used too.
The end result is multiple identical copies of the
same human DNA segment or gene.
Benefits of DNA cloning
Cloning produces enough copies of a gene or DNA
segment to be analysed for human genetic testing
or can be used for the development of drugs and
treatments for genetic conditions.

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Figure 22.1: By fragmenting DNA of any origin (human,
animal, or plant) and inserting it in the DNA of rapidly
reproducing foreign cells, billions of copies of a single gene
or DNA segment can be produced in a very short time. The
DNA fragment of interest from an organism such as a human
is incorporated into the ‘plasmid DNA’ of a bacterial cell
Figure courtesy of https://public.ornl.gov/site/gallery/
detail.cfm?
id=385&topic=&citation=&general=cloning&restsection=all)
(b) Reproductive Cloning or adult DNA Cloning
Reproductive cloning is also called adult DNA
cloning. The purpose of this type of cloning is to
produce a genetic duplicate of an existing or
previously existing organism.
How reproductive cloning is done
Dolly the cloned sheep produced in 1997 is an
example of an animal produced by cloning as
shown in Figure 22.2. She was produced in a
process called somatic cell nuclear transfer
(SCNT).

A somatic cell is any cell in the body other than
the sperm or egg reproductive cells.
In SCNT, the nucleus of a somatic cell isolated
from the donor animal is put in an egg cell that
has had its nucleus removed. The egg cell is
treated with an electric charge or chemicals to
simulate fertilisation and cell division to produce
an embryo.
The newly formed embryo can be implanted into a
surrogate mother and carried to term. An embryo
created by SCNT is a genetic replica of the donor
animal.
The difference between an embryo created by
SCNT versus an embryo created naturally is where
the two sets of chromosomes that make up the
embryo came from. We normally have 23 pairs of
chromosomes.
We receive 1 of each pair from our mother and 1
of each pair from our father. In SCNT, all of the
chromosomes come from a somatic cell from the
donor. Thus the cloned embryo receives all of the
chromosomes from one adult.
An important step in the development of the
cloning technology of whole animals is being able
to activate the genes that are needed for an
embryo to grow and develop that are inactivated
soon after their job has been done. Adult cells
therefore have to have these genes ‘turned on’
again.

The discovery of how to do this embryonic gene
activation enabled the cloning of Dolly and the
technique has since been replicated with a
number of other animals including mice, pigs,
goats, cats, rabbits and cows.
It should, however, be noted that a clone such as
Dolly produced by SCNT is not an exact replica of
the donor animal.
This is because while Dolly has the same
chromosomal DNA located in the nucleus as the
white-faced donor sheep, the clone’s
mitochondrial DNA comes from the egg of
another animal – in this case a black-faced sheep.
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Figure 22.2: Cloning of Dolly step by step.
1: Skin cells removed from the udder of a white faced Finn
Dorset Ewe and placed in a growth culture containing very
low nutrients: the cells are starved; stop dividing and the
active genes are switched off.
2: The DNA is removed from an egg cell from a Scottish
Blackface Ewe leaving the egg cell cytoplasm containing
mitochondria.
3: The skin cell and the egg cell are placed next to each
other and subjected to an electric pulse to cause fusion. The
fused cell contains 46 chromosomes from the Finn Dorset

Ewe in the cytoplasm of the Scottish Blackface Ewe. The
fused cell is given a 2nd electric pulse to activate cell division
and turn on the genes necessary for growth of the embryo.
4: The embryo is then implanted into the uterus of a
different Scottish Blackface Ewe. Dolly, a coned white faced
Finn Dorset lamb, is born.
Figure courtesy of: http://science.howstuffworks.com/
cloning2.htm
Chromosomes are found in the nucleus of all
body cells except for red blood cells which have
no nucleus and therefore do not contain
chromosomes.
Mitochondrial DNA is found is in very small
compartments called mitochondria (the energy
centres of the cell) that are found scattered
outside the nucleus (Figure 22.3). Mitochondrial
DNA has its own genes, arranged as one long
circle of DNA.
Benefits and limitations of reproductive cloning
Reproductive cloning can have many uses:
could be improved as discussed below, the
technology can be used to mass-produce
animals with special qualities, such as animals
that are important agriculturally or are able
to produce helpful drugs for human use
used to repopulate endangered species, as
has been shown with the wild ox and the gaur

cloning also see it as a way of overcoming
male infertility, where other methods of
assisted reproduction have failed.
The difficulties however in producing cloned
animals are reflected in the number of
abnormalities seen in the experiments done to
date.
not viable: only one or two offspring are
viable for every 100 cloning attempts
gher rates of cancer
and infection, and a range of other health
problems
problems often have appeared later in the life
of the resulting clone, so that mature clones
have often undergone sudden, unforeseen
and unexplainable deaths.
One of the reasons for the high rates of death
and abnormalities in cloned animals is perhaps in
the reprogramming of the genetic make-up from
an adult cell into an embryonic cell that must take
place. Some genes will only be switched on or off
if they are passed to an embryo through the egg
or sperm. This is called imprinting.

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Figure 22.3: Diagram of a human cell showing nuclear DNA
which is found on chromosomes in the nucleus of a
cell and the mitochondrial DNA which is found in the energy
centres of cells known as mitochondria. Figure
adapted from the NHS National Genetics and Genomics
Education Centre.
Ethical considerations in reproductive cloning
Human reproductive cloning has raised many
concerns, not the least of which are the safety
considerations and difficulties listed above.
While these technical difficulties may be
overcome in the future and may enable cloning of
animals with a greater success rate, the use of
reproductive cloning for humans is viewed
differently.
essential for human growth and health. It is
also important to realise that a person is
much more than a product of their genes.
human, the clone would not be a duplicate of
that person, other than the nuclear DNA.
A person is a product of their environment as

well as their genetic make-up. Even identical
twins have subtle differences between them.
of social responsibilities are all challenged by
human reproductive cloning.
(c) Therapeutic cloning to produce stems cells
The aim of therapeutic cloning is to create stem
cells containing the person’s own DNA that could
be grown in the laboratory and then transplanted
into them without the risk of tissue rejection.
Diseases that might be treated by transplanting
cells generated from human stem cells include
Parkinson disease, diabetes, traumatic spinal cord
injury, Duchenne muscular dystrophy, heart
disease, and vision and hearing loss.
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Therapeutic cloning using embryos
When using embryos as the source of the stem
cells, this type of cloning is also known as
therapeutic cloning, biomedical cloning, embryo
cloning or artificial embryo twinning.
This type of cloning essentially mimics the natural

process of producing identical twins or triplets.
egg tries to divide into a two-cell stage but
the two cells separate instead
their own and eventually develop into two
genetically identical individuals.
In embryonic cloning, cells are removed from an
embryo after it has divided for about 5 days. At
this stage, the embryo is called a blastocyst. Once
the cells are removed the embryo is destroyed.
The cells of an embryo are an excellent source of
stem cells because they have not yet been
differentiated into the cells of specific tissues or
organs of the body. While there are around
20,000 genes in each body cell, only those genes
that are needed for the function of the cells of
the tissue or organ are switched on. The
remainder are inactive.
For example, brain cells will have different genes
sending instructions to the cells than liver cells.
Embryonic stem cells still have all the genes
needed for the function of the body’s cells to be
able to be switched on and so can be
differentiated into any type of body cell.
To generate cultures of specific types of
differentiated cells — heart muscle cells, blood
cells, or nerve cells, for example — scientists try
to control the differentiation of embryonic stem
cells.

They change the chemical composition of the
culture medium, alter the surface of the culture
dish, or modify the cells by inserting specific
genes. Through years of experimentation
scientists have established some basic protocols
or recipes for the directed differentiation of
embryonic stem cells into some specific cell types.
Therapeutic cloning using adult cells
An alternative source of stem cells are adult stem
cells that can be extracted from adult tissue such
as the bone marrow or fat tissue without harm to
the individual.
found among differentiated cells in a tissue or
organ. It can renew itself and can
differentiate to yield the major specialised cell
types of the tissue or organ
dult stem cells in a
living organism are to maintain and repair the
tissue in which they are located
instead of adult stem cell
defined by their origin (the inner cell mass of
the blastocyst), the origin of adult stem cells
in mature tissues is unknown.
Adult stem cells occur in many more tissues than

was once thought possible, and adult stem cells
from bone marrow that form into mature blood
cells have been used in transplants for 30 years.
Tissues include brain, bone marrow, peripheral
blood, blood vessels, skeletal muscle, skin and
liver.
There are, however, often only a very small
number of stem cells in each tissue. Stem cells are
thought to reside in a specific area of each tissue
where they may remain quiescent (non-dividing)
for many years until they are activated by disease
or tissue injury.
Certain kinds of adult stem cells seem to have the
ability to differentiate into a number of different
cell types, given the right conditions. If this
differentiation of adult stem cells can be
controlled in the laboratory, these cells may
become the basis of therapies for many serious
common diseases.
For example adult stem cells can be used in the
treatment of type 1 diabetes where the damaged
pancreatic cells that produce insulin are replaced,
removing the requirement for daily administered
insulin (Figure 22.4).
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Figure 22.4: A doctor takes a sample of skin cells from the
patient and isolates their DNA. Next, a donor egg cell, emptied
of its
own genetic contents, is injected with the DNA from the
patient. The embryo is nurtured to grow and divide into a
blastocyst.
Some blastocyst cells are harvested and coaxed with growth
factors to mature into insulin-producing cells. Finally, millions
of
insulin-producing cells are injected back into the patient. In an
ideal world, the patient’s diabetes is temporarily ‘reversed’,
with no side effects. Figure adapted from: Stem Cells: A
Primer, US National Institutes of Health- Source: Cloning
around with
stem cells http://abc.net.au/science/slab/stemcells/default.htm
Benefits and limitations of therapeutic cloning
Whatever their source, before stem cells can be
used to treat health problems, a number of
barriers have to be overcome.
from a source and then grown in the
laboratory
into the specific cell type needed for
treatment.
These first two hurdles have been passed for
most of the 220 cell types in the human body.
Overcoming the next barriers of applying the
technology clinically and ensuring that the new
tissue or organ poses no risk to the patient is still

a matter of much research.
While one organ, the skin, is already able to be
grown in the laboratory to create a self-
compatible skin graft, skin is relatively easy to
grow because the mature, differentiated skin cells
are still able to divide and produce more cells to
repair damage. The cells of other organs or
tissues do not have this ability.
An alternative is to create genetically modified
pigs from which organs suitable for human
transplantation could be harvested.
Pig tissues and organs are the most similar to
humans of the animal species that have been
cloned. The transplant of organs and tissues from
animals to humans is called xenotransplantation.
One of the major concerns, however, in the
transplant of pig tissues and organs to humans, is
the transmission of pig viruses to humans.
While adult stem cells may overcome some of the
ethical concerns, further studies are needed to
determine their ability to differentiate into a
variety of new types of cells.
Ethical considerations in therapeutic cloning
Cloning presents many complex ethical questions.
A few to consider are who should have access to
the technologies, how will the use of the
technologies be monitored, who gets to decide
what aspects of cloning are morally acceptable
and legal to use and which are not. So even if it is
possible to clone humans – should we?

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The therapeutic cloning technology used to
harvest stem cells ultimately results in the
destruction of an embryo. The creation of
embryos specifically for the purpose of destroying
them causes concern for some people.
The advantage of using adult cells as a source of
stem cells is that it would avoid this ethical issue
of destruction of the embryo.
Currently, embryos that are used for the
production of stem cells are those created by
infertile couples during in vitro fertilisation (IVF)
programs. Usually more embryos than are
needed to have a baby are produced. Some
couples have agreed for their excess embryos to
be used for stem cell research.
CLONING POLICIES IN AUSTRALIA
In 1999, the Council of the Australian Academy of
Science unanimously endorsed a position
statement called ‘On Human Cloning’ (https://
www.science.org.au/sites/default/files/user-
content/clone.pdf) that recommended a ban on
human reproductive cloning but recognised the
potential of therapeutic cloning and considered
that it should be fostered in research.

Australia has passed legislation banning human
reproductive cloning (Prohibition of Human
Cloning for Reproduction and the regulation of
Human Embryo Research Amendment Act 2006)
but has treated therapeutic cloning differently by
passing the Research Involving Human Embryos
Act 2002.
is Act was to address
concerns, including ethical concerns, about
scientific developments in relation to human
reproduction and the utilisation of human
embryos by regulating activities that involve
the use of certain human embryos created by
assisted reproductive technology (ART)
(created after April 5 2002 by ART with the
aim of achieving a pregnancy) that are in
excess of a couple’s needs, and with their
informed consent, for stem cell research. This
approval applies when that research is
governed and overseen by a committee of the
National Health and Medical Research
Committee (NHMRC)
embryos through therapeutic cloning and
extract their stem cells for use in medical
research.
Currently, therapeutic cloning research is being
conducted in private laboratories in the USA and
in both government and private laboratories in
the UK, Japan, France, Australia, and other

countries.
G e n e t i c s , t e c h n o l o G y , a n d s o c i e t y
stem cell Wars
Stem cell research may be the most controversial research area
since the beginning of recombinant
DNA technology in the 1970s. Although
stem cell research is the focus of presiden-
tial proclamations, media campaigns, and
ethical debates, few people understand it
sufficiently to evaluate its pros and cons.
Stem cells are primitive cells that rep-
licate indefinitely and have the capacity
to differentiate into cells with special-
ized functions, such as the cells of heart,
brain, liver, and muscle tissue. All the
cells that make up the approximately 200
distinct types of tissues in our bodies are
descended from stem cells. Some types of
stem cells are defined as totipotent, mean-
ing that they have the ability to differenti-
ate into any mature cell type in the body.
Other types of stem cells are pluripotent,
meaning that they are able to differentiate
into any of a smaller number of mature
cell types. In contrast, mature, fully differ-
entiated cells do not replicate or undergo
transformations into different cell types.

In the last few years, several research
teams have isolated and cultured human
pluripotent stem cells. These cells remain
undifferentiated and grow indefinitely in
culture dishes. When treated with growth
factors or hormones, these pluripotent
stem cells differentiate into cells that
have the characteristics of neural, bone,
kidney, liver, heart, or pancreatic cells.
The fact that pluripotent stem cells
grow prolifically in culture and differen-
tiate into more specialized cells has cre-
ated great excitement. Some foresee a
day when stem cells may be a cornucopia
from which to harvest unlimited numbers
of specialized cells to replace cells in dam-
aged and diseased tissues. Hence, stem
cells could be used to treat Parkinson dis-
ease, type 1 diabetes, chronic heart dis-
ease, Alzheimer disease, and spinal cord
injuries. Some predict that stem cells will
be genetically modified to eliminate trans-
plant rejection or to deliver specific gene
products, thereby correcting genetic de-
fects or treating cancers. The excitement
about stem cell therapies has been fueled
by reports of dramatically successful ex-
periments in animals. For example, mice
with spinal cord injuries regained their
mobility and bowel and bladder control
after they were injected with human stem
cells. Both proponents and critics of stem
cell research agree that stem cell therapies

could be revolutionary. Why, then, should
stem cell research be so contentious?
The answer to that question lies in the
source of the pluripotent stem cells. Until
recently, all pluripotent stem cell lines were
derived from five-day-old embryonic blas-
tocysts. Blastocysts at this stage consist of
50–150 cells, most of which will develop into
placental and supporting tissues for the early
embryo. The inner cell mass of the blastocyst
consists of about 30 to 40 pluripotent stem
cells that can develop into all the embryo’s
tissues. In vitro fertilization clinics grow fer-
tilized eggs to the five-day blastocyst stage
prior to uterine transfer. Embryonic stem
cell (ESC) lines are created by taking the in-
ner cell mass out of five-day blastocysts and
growing the cells in culture dishes.
The fact that early embryos are de-
stroyed in the process of establishing
human ESC lines disturbs people who
believe that preimplantation embryos
are persons with rights; however, it does
not disturb people who believe that these
embryos are too primitive to have the
status of a human being. Both sides in
the debate invoke fundamental ques-
tions of what constitutes a human being.
Recently, scientists have developed
several types of pluripotent stem cells
without using embryos. One of the most
promising types—known as induced plu-
ripotent stem (iPS) cells—uses adult somatic

cells as the source of pluripotent stem cell
lines. To prepare iPS cells, scientists iso-
late somatic cells (such as cells from skin)
and infect them with engineered retrovi-
ruses that integrate into the cells’ DNA.
These retroviruses contain several cloned
human genes that encode products re-
sponsible for converting the somatic cells
into immortal, pluripotent stem cells.
The development of iPS cell lines has
generated renewed enthusiasm for stem cell
research, as these cells bypass the ethical
problems associated with the use of human
embryos. In addition, they may become
sources of patient-specific pluripotent stem
cell lines that can be used for transplanta-
tion, without immune system rejection.
At the present time, it is unknown
whether stem cells of any type will be as
miraculous as predicted; however, if stem
cell research progresses at its current rap-
id pace, we won’t have long to wait.
Your Turn
Take time, individually or in groups, to answer the following
questions. Investigate the references and links
to help you understand the technologies
and controversies surrounding stem cell
research.
1. What, in your opinion, are the scien-
tific and ethical problems that still
surround stem cell research? Are these

problems solved by the new methods
of creating pluripotent stem cells?
You can find descriptions of some new methods
of generating pluripotent stem cell lines, and
the ethical issues that accompany these meth-
ods, in Kastenberg, Z. J. and Odorico, J. S.
2008. Alternative sources of pluripotency: sci-
ence, ethics, and stem cells. Transplantation
Rev.22: 215–222.
2. What are the current stem cell re-
search laws in your region, and how
do these laws compare with national
regulations?
A starting point for information about stem cell
research regulations can be found on the Stem
Cell Information Web site of the National Insti-
tutes of Health (http://stemcells.nih.gov).
3. Do you oppose or support stem cell
research? Why, or why not?
An interesting online poll, along with argu-
ments for and against stem cell research, is
offered by the Public Broadcasting Corpora-
tion, at http://www.pbs.org/wgbh/nova/
body/stem-cell-poll.html.
4. What, in your opinion, is the most
significant development in stem cell
research in the last year?
Some ideas to start your search are: the PubMed
Web site (http://www.ncbi.nlm.nih.gov/

sites/entrez? db=PubMed) and the New
York Times online Stem Cell page (http://
topics.nytimes.com/top/news/health/
diseasesconditionsandhealthtopics/
stemcells).

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