Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
Cloning
Project
Project work
work
"The cloning of humans is on most of the lists of things to worry about from Science, along with
behavior control, genetic engineering, transplanted heads, computer poetry and the
"But we realize that with any new and powerful technology with unknown, and to some degree
unrestrained growth of plastic flowers."
unknowable - by definition - effects, then there necessarily will be an appropriate level at least,
and maybe even more than that, of public debate and public interest."Bob Shapiro
Lewis Thomas
February 2014
Mentors: prof. Sonja Artač, prof. Maja Gerden
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
Table of contents
1.
Summary ............................................................................................................................ 3
2.
Introduction........................................................................................................................ 4
3.
History of cloning ............................................................................................................... 5
4.
Branches of cloning ............................................................................................................ 7
Reproductive cloning.............................................................................................................. 7
Therapeutic cloning ................................................................................................................ 7
5.
Techniques for the cloning of cells..................................................................................... 8
Roslin technique ..................................................................................................................... 8
Artificial Embryo Twinning ..................................................................................................... 9
Honolulu technique ................................................................................................................ 9
6.
Molecular cloning ............................................................................................................. 10
Procedure of molecular cloning ........................................................................................... 10
7.
Genetically modified organisms (GMO) ........................................................................... 12
Can we choose? .................................................................................................................... 12
8.
Opportunities, pitfalls and problems of cloning .............................................................. 13
Opportunities ................................................................................................................... 13
Pitfalls ............................................................................................................................... 14
Problems........................................................................................................................... 14
9.
10.
Conclusion ........................................................................................................................ 16
Sources and literature ................................................................................................... 17
Images
Image 1 – Therapeutic cloning ................................................................................................... 7
Image 2 – Roslin technique ........................................................................................................ 8
Image 3 – Molecular cloning .................................................................................................... 11
Image 4 – GSO .......................................................................................................................... 12
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
1. Summary
We decided on the topic “Cloning”. We chose this topic because it interested us and we
don’t hear much about it in our daily lives.
We mostly focused on cloning techniques, the pros and cons of cloning, the procedure itself,
where we use it, different types of cloning and consequences that may occur on a living
being that was a part of these tests. However we didn’t talk about ethics of cloning because
we are interested in facts that’s why we purposely avoided this and focused on “how”,
rather than “but, what if” or “is this right?”
In our project work we’ll be mentioning cloning techniques such as: Roslins technique,
artificial embryo twinning and the Honolulu technique.
We also researched some things about GMO (Genetically modified organisms), how they
affect our daily lives and how they’re displayed in the public.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
2. Introduction
Each day scientists make progress and find new and more effective ways on how to clone a
living being. We all know the sheep called 'Dolly'. She was the first cloned mammal, but not
many people know what the procedure to clone her was like and in which try they got it
right.
Of course cloning didn’t begin with Dolly. Cloning had been around for at least thousand
years. For example: 'Pando' is a group of trees around Fish Lake in Utah – US. They all have
the same root system, which means they are all a single tree. This tree is about 80.000 years
old and is one of the oldest living beings on planet Earth. Meaning, all these 'trees' are
clones, since they're genetically exact copies of each other.
Another example of clones is an earthworm. If they are somehow cut in half, both halves can
survive and regenerate all missing parts, hence creating two genetically same earthworms.
Us humans have always tried being better than mother nature. In 19th century Hans Dreich
wanted to show that during cell division the genetic material isn't lost. While doing this he
actually created two identical living beings – making him the 'founder' of modern day
cloning.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
3. History of cloning
Cloning is the process of creating an identic copy. In a biologic meaning this can be a
molecule, a cell, tissue or a multicellular organism. Clones may arise in a natural way, for
example: asexual reproduction. However this word is more commonly used to describe
identical copies which have been created intentionally.
History of cloning began in 1885 with a demonstration of duplication of an artificial sea
urchin embryo.
Hans Adolf Edward Dreisch separated a two-cell embryo of a sea urchin. Both cells produced
a living adult organism. With this experiment he proved, that each cell contains full
hereditary information that is needed for a successful development of the organism.
Hans Spemann tried to do the same with an embryo of a salamander but to split the two
cells used a loop made from a baby’s hair. This embryo developed successfully into an adult
organism. He also found out that it is harder to separate more developed embryos and that
these do not develop as well as they could’ve if they had been separated sooner.
In his second attempt, Spemann, with the help of a baby’s hair squeezed the fertilized egg
and doing so pushed the nucleus towards the outer membrane. Later only the part of the
zygote containing the nucleus devided. After some time, he moved one of the nuclei that has
successfully duplicated into the part that had no nucleus and separated them afterwards.
Both embryos developed successfully.
This Spemann’s experiment was the first case of transporting the nucleus; however the first
official transport was done by Robert Briggs and Thomas King. They took the nucleus of a
tadpole embryo and transferred it into a frog’s egg, from which they have previously
removed the nucleus. A tadpole successfully developed from this cell soon after. This
experiment proved that the transportation of the nucleus is a doable technique,
furthermore they also proved that the cells of an embryo in the early stages of development
are more likely to produce wanted results that those which have already gone through some
stages of development.
Jonh Gurdon went ever further. He took the nucleus of a cell that was part of the intestine
of a tadpole and transferred it into a frog’s egg and doing so created two genetically equal
tadpoles. With this experiment he proved that the hereditary informations inside the
nucleus stays the same throughout division and differentiation.
1975 was the year in which the first embryo of a mammal was created using the later
mentioned ‘Roslin technique’. They took the nucleus of a rabbit embryo cell and moved it
inside a rabbits egg. This successfully developed firstly into a morula1 and later into an
embryo.
1
A morula is an embryo at an early stage of embryonic development.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
Steen Willadsen separated a cell out of an eight cell lamb embryo. With a mild electro shock
he fused the egg which he previously removed with this cell and the newly created cell
began to divide. He cultivated the cells in vitro2 and later moved the embryo into a
substitute mother which gave birth to three living lambs.
2
Processes and experiments that take place inside a controlled environment. This process in opposite to ‘in
vivo'. The method 'in vitro' was already used as a mean of artificial fertilization.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
4. Branches of cloning
There are two branches of cloning. We choose from these two depending on the objective to
be achieved.
Reproductive cloning
Reproductive cloning is used when we want to create two genetically equal organisms, while
at least one of them is already living. The nucleus of an adult cell of the donor is transported
into the egg whose nucleus has been taken out previously, meaning it has no hereditary
information. After this is done the egg starts to divide and when it reaches a certain point it
is transferred into the womb of a substitute mother.
Therapeutic cloning
Therapeutic cloning is used as a mean of creating stem cells which can be used for research
of the human body and medical reasons, such as treatment of a disease. Stem cells can be
acquired from a cloned embryo old approx. 5 – 6 days (blastocyst). This can be cultivated in
vitro.
Image 1 – Therapeutic cloning
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
5. Techniques for the cloning of cells
Roslin technique
This method is based on transport or exchange of the cell nucleus.
Sheep Dolly was the first mammal that has been successfully cloned (out of 277 tries) from
an already differentiated body cell. Cloning was done by Ian Wilmut and Keith Campbell
with co-workers from Roslins
institute that is a part of University
in Edinburgh. Dolly was cloned
with so-called ‘Roslin technique’.
They took the egg of a sheep
(breed ‘Scottish Blackface’) and the
body cell of a mammary gland,
again from a sheep (breed ‘Finnish
Dorset’). Somatic cells were
treated in a lab in such a way, that
they became similar to
undifferentiated stem cells. They
became totipotent3 respectively
pluripotent4. They removed the
nucleus from the egg and inserted
the nucleus from the mammary
gland which contains high numbers
of necessary proteins that the
embryo needs to survive.
These cells; with the help of mild
electro shocks, started dividing and
these embryos were later inserted
into a substitute mother. Sheep
Dolly was born on 5th of July 1996
and attracted a lot of media
attention.
Image 2 – Roslin technique
3
4
Totipotent stem cells are those that can differentiate into all kind of cells.
Pluripotent refers to a stem cell that has the potential to differentiate into any of the three germ layers
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
Artificial Embryo Twinning
This technique is similar to the natural process that makes the cell divide in the way that it
produces twins. With twins, after the first division the cells of the fertilized egg separate and
evolve separately creating two genetically equal organisms.
Honolulu technique
This technique is widely compared to the Roslin technique, but it is believed to be better.
In 1998 a group of scientists with Terihukom Wakayamo and Ryuzom Yanagimachijem as
head scientists successfully cloned three generation of genetically identical house mice. Mice
were hard to clone for a long time because the division of the egg starts right after
fertilization and there is no time to reprogram the nucleus. Out of one hundred tries only
three were successful.
In Honolulu technique supporting cells were used in the testes (seritoli cells), nerve cells and
granulose cells that form granulated fund around oocytes (cumulus cells), and they are
almost always in the G0 phase.
A nucleus was removed from the mice cell and transferred into the egg. After an hour, the
egg adopted the new nucleus, the cell was moved to the cell culture in which it began to
grow, divide and develop. Cytochalasin B was added to the culture, which prevents that
during the division a polar body would be created, which would result in the formation of
haploid cells. The cell was successfully developed into an embryo, which was inserted into
the substitute mother.
Out of all three types of cell which this technique was tested on, the most promising was
cloning of granulose cells.
Wakayama later created clones of clones and let them reproduce among one another. This
showed their reproduction went on with no problems and that clones of clones could
reproduce without difficulties.
Compared to the Roslin technique the Honolulu yields much better results (50:1).
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
6. Molecular cloning
Molecular cloning is an experimental method that makes the creation of recombined DNA
molecules possible. With it, it’s also possible to direct the duplications of DNA. In comparison
to previously mentioned techniques, this is not used to clone whole cells but it is used to
create an organism that contains a part of foreign DNA. We call them transgenes or
genetically modified organisms (GMO).
Procedure of molecular cloning
Choosing the host organism and the cloned vector5
For the host organism bacteria is the number one choice. For example: the bacteria
Echerichia coli which are benign6 and easy to cultivate. The vector party is most often
represented by a plasmid or a bacteriophage. Of course other combinations also exist, since
the choice host-vector depends on the combination of organisms between which the
transfer of genes happens.
The vector almost always contains 4 key sections of DNA:




place where the duplications happens and enables the vector to duplicate inside the
host organism,
area of cleavage site where foreign DNA is inserted,
a marker gene, which allows the survival of those host cells which have adopted the
vector, as it is needed to separate them from those that have not adopted the vector
for any further processing,
at times even an additional gene, which serves as an identifier of those cells that
have also adopted the foreign DNA so later those cells can be used to produce
transgenic organisms.
Preparation of vector DNA
With the help of enzymes the vector DNA is split at the place where the foreign DNA is to be
inserted. It is split where the building vector DNA molecules are uniquely determined and in
this way they prevent the DNA vector to cleave in the wrong place.
Preparation of DNA that is to be cloned
DNA can be acquired from whichever tissue, even from tissues of extinct animals. They
cleave it so they get fragments that can be attached to ends of the vector.
Production of recombinant7 DNA
The fragments of vector DNA and the DNA of a foreign source is mixed with the enzyme
DNA-ligase, which bounds the fragments between one another in DNA that is made up of
sections of different origin. This process is called ligation.
5
DNA section that make the transfer of foreign hereditary information into the host cell possible
Harmless
7
DNA is made up of sections of different origin.
6
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
Entry of recombinant DNA into the host organism
Recombinant DNA is inserted into the host organism. This process is not very effective
because only a small amount of organisms accepts the recombinant DNA.
For the transfer of the recombinant DNA, again 4 methods can be used but they depend on
the type of organism:




transformation - the transfer of a naked DNA through the cell membrane, wherein
the cells are not in contact with one another; typical for bacteria,
transfection – this process is similar to transformation, but with mammals,
electroporation - with the help of high-voltage electrical pulses, useful for plants and
animals,
transduction - DNA is introduced into the cell with the help of structures, similar to
viruses.
Choosing organisms that have adopted the recombinant DNA
For the separation of those organisms that have adopted the recombinant DNA from those
who didn’t, artificial genetic selection is normally used. All organisms are exposed to
conditions (e.g. antibiotics or environment without essential substances), in which only
those organisms can survive that have adopted the vector sequence and with that the gene
that enables them to survive in such conditions.
Searching for clones with the wanted biological properties
This phase is time-consuming because it is necessary to examine a large number of different
clones and make sure that they are really getting the organism with the desired properties.
Such organisms are then left to proliferate.
Image 3 – Molecular cloning
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
7. Genetically modified organisms (GMO)
In the natural process of sexual reproduction new organisms are produced with
recombination of genes; sexual cells contain these. Today, under the "GM" tag we classify
any organism that carries a novel combination of genes that are acquired through use of
modern methods of biotechnology.
Can we choose?
The development of genetic engineering enabled us the transfer of genetic material among
those organisms which naturally cannot exchange their genes. Opinions regarding the use of
GMOs are different. Some experts believe such interventions are harmless to humans and
the environment, while others are worried about what side effects transferring genes from
one organism to another may have. The most common GMOs, which are used in the food
industry:





corn,
soy,
rapeseed,
potato,
tomato.
Because not all effects of GM food are yet known, many countries started thinking about
labelling such food to give the option of decision to the consumer.
Countries in which labelling GM food is obligatory:





Japan (April 2001)
Philippines (August 2001)
EU (April 2000)
Switzerland (2002)
Australia, New Zealand (July 2001)
Image 4 – GSO
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
8. Opportunities, pitfalls and problems of cloning
Opportunities
Reproductive cloning
•
With reproductive cloning we could create a genetically connected child, who
would have genes that belong to both parents even if the parents were infertile,
homosexual or lost their child and wanted a genetically identical copy.
•
With animals and plants this could be used to prevent extinction. In 2005 north
Korean scientist already cloned 2 specimen of an endangered species of wolf,
who could be viewed and studied by the public in a local zoo.
Therapeutic cloning
•
With therapeutic cloning scientist can create tissue and organs in a way where
they take healthy cells of the patient and create a lot of copies of those cells and
with them they fix the damaged or diseased tissue or organ.
•
There are organisations such as banks of stem cells, where an individual can
donate his stem cells, for example stem cells of bone marrow. These can be used
to cure cancer of the individual or a person with similar genetic predisposition.
•
Scientists have not yet been able to create a whole organ out of stem cells,
however in a American university in Pittsburg heart tissue has already been made
and this tissue started spontaneously contracting. Heart stem cells were made
from pluripotent skin cells (which were in G0 phase). These cells were transferred
onto the frame of the mouse heart whose cells were previously removed. After
20 days of growth and blood supply the cells reconstructed the mouse organ and
started contracting at 40-50 beats per minute. This was a great achievement even
though a mouse heart with human heart cells can’t help us very much at this
point.
Molecular cloning
•
In somatic cells we can change defective genes with functional ones and doing so
we can treat genetic disorders, illnesses and defective tissues, which can later be
returned back to the patient. We can also change sexual cells and with this the
whole organism and consequential all later generations.
•
Molecular cloning enabled us the overview of the entire DNA structure, which
makes studying separate genes possible. With molecular cloning they created
proteins and with these proteins we can fix defective or poorly expressed genes.
They are also useful when treating illnesses, (e.g. stroke). They make the
production of vaccine that stops infectious diseases from spreading possible.
•
Transgene animals (cows, sheep…) which have been inserted with a human gene
can be used as a source of pure human protein, e.g. insulin.
•
If sexual cells are genetically modified the whole organism can be changed
respectively all later generations. Organisms that are modified in such a way are
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
•
today mostly used in the food and pharmaceutical industry but also more and
more in agriculture.
Transgene fish have a jellyfish gene in their DNA and this gene makes them emit a
fluorescent colour that makes them glow in the dark. In south China glowing pigs
were created. These organisms wouldn’t naturally survive but they represent a
diversification in the choice of pets.
Pitfalls
Reproductive cloning
•
From previous experience we learned that with cloning there are many mistakes.
Wilmut suggested that to successfully create a human clone we would need
about 1000 egg cells. (CITAT)
•
Sheep Dolly died from lung illness and very bad arthritis. Sheep of the same breed
have an average life span from 11 to 12 years. Dolly died at 6 and a half. They
discovered that telomeres which shorten when DNA duplicates were shorter and
that indicates that Dolly was 6 years older when she was born, the same age as
the sheep who donated the mammary gland.
Therapeutic cloning
•
Even though therapeutic cloning helps with progress in medicine and science an
egg with a donor nucleus can be very unstable. For a single stable egg, even up to
100 experiments are needed.
Molecular cloning
•
GM plants can infect other non-GM plants with their pollen even those over a
kilometre away. If this happens the genuine “purity” of species can be lost.
Ecologic agriculture with GM plants present in the fields is not possible.
•
Research in Jena has shown that bees that were collecting nectar on a nearby
farm of GM oil seed rape had a change in their intestinal flora which has a
negative effect on the development and life of a family of bees. The French
government institute INRA found out that the pollen of a GM oil seed rape and
soy shortens the average bee life span and affects their behaviour.
•
In addition to concerns about the environmental impact, experts are also
concerned about unforeseen side effects, which artificially transferring genes
from one organism to another may have on human health. The consequences of
long-term use of GMOs not yet known. There is also likelihood that GMOs
increase the number of allergic diseases. Due to the fear of an increase in allergic
reactions, for example, were not allowed to insert a Brazil nut gene into soybean.
Problems
Lots of times we hear that scientist have cloned a new animal even though they had
problems before, meaning science is experiencing progress in this field. However a lot of
times they forget to mention how successful this was and in how many tries they got it.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
It is true that the success of cloning is increasing, but it’s still low, as the potential for errors
is high because methods of cloning are complex. The very first may be that the nucleus and
the egg may be incompatible and the egg will reject nucleus rejected. Perhaps the zygote
isn’t dividing and developing or the fetus may die in a substitute mother, also an error may
occur in the process of cloning.
If older genetic material is to be used and we transfer it into an egg new cells are created
that take up all the errors in the genetic code of the previous cell. As we previously stated,
this happened with Dolly (shorter telomeres), however it is true that shortening of the
telomeres is not typical for this particular breed.
Expressing genes is one of the main problems with clones. A lot of times there express
differently than they should’ve and as consequence the clone phenotypically8 isn’t as it is
expected and that can cause serious medical problems (e.g. cancer). Clones are typically
larger than those that are created naturally. Especially with in vitro fertilization the LOS
(Large offspring syndrome) arises. This happens because the fibroblast growth receptor
(FGFR2) is deactivated consequentially the embryo growth is sped up. These clones have
organs that are bigger organs than their body can handle meaning they have problems with
enough blood reaching and supplying all organs, breathing and other life processes.
8
Part of genes that are expressed, unlike the genotype which is all genes even those that weren't expressed.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
9. Conclusion
Cloning has a bunch of positive sides but as everything on planet earth this also has negative
effects. We can always suggest that thing won't get out of hand. Even the smallest mistake
can have an impact on the outcome. With microscopic cloning present and in use, we can
almost say that scientists are creating DNA out of Lego blocks.
On one hand we can prevent hunger and make life better on a global scale, while on the
other hand we don't know who and when will open the Pandora’s box and create an
organism that may be the end of us.
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
10. Sources and literature
Books
Agar, N.; Popolna kopija: razpletanje razprave o kloniranju, Ljubljana, Založba Krtina, 2008,
159 str., str. 25-95, ISBN: 978-961-260-007-5
Gilbert, F. S., Tyler, A. L., Zackin, E. J.; Bioetika in sodobna embriologija : izhodišča za
razpravo, (prevod Mina Mušinović, Mira Hladnik, Tamara Mušinović Zadravec), Podsmreka,
Pipinova knjiga, str. 113-126, 131, 2013, ISBN 978-961-93354-3-7
Stušek, P., Vilhar, P.; Biologija celice in genetika: učbenik za biologijo v programih
gimazijskega izobraževanja (Ljubljana, DZS, 2010) str. 323, 324, 326
Dermastia, M., Komel, R., Turk, T.; Kjer se življenje začne ---: biologija celice in genetika za
gimnazije (Ljubljana, Rokus Klett, 2011) str. 234-242
Internet articles and other sources from the internet
-
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
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Wikipedia, Molekulsko kloniranje, URL http://sl.wikipedia.org/wiki/Molekulsko_kloniranje
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Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
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Aligator , URL
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~ 20 ~
Izak Oberčkal Pluško, Jan Slunečko: Cloning (project work for BIOP)
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