GENE MANIPULATION

WOLLY MAMMOTH
The woolly mammoth (Mammuthus primigenius) was a species of mammoth that lived during
the Pleistocene epoch; it was one of last in a line of mammoth species. Woolly mammoths are
closely related to this day and ages Asian elephants but died out in the last Ice Age. The last of the
species survived on Wrangel Island in the Arctic Ocean until about 3,300 years ago.
The woolly mammoth was coarsely the same size as modern African elephants. Males reached
heights between 2.7 and 3.4 m weighed up to 6 tonnes. Females were around 2.62.9 metres in
height and weighed up to 4 tonnes. A new-born calf weighed about 90 kilograms.
The woolly mammoth was well adapted to handle the harsh cold environment it was subjected to in
the last ice age. It was covered in fur, armouring an outer covering of long guard hairs and a shorter
undercoat. The ears and tail were short to minimise frostbite and heat loss, as externalities of the
body usually get frost bite first.
The woolly mammoth has behaviour that was quite similar to that of todays elephants, as it also
used its tusks and trunk for working objects, fighting, and scavenging. The diet of the woolly
mammoth consisted of mainly grass and sedges (a type of bushy grass). Individuals could probably
reach the age of 60 years old. Its habitat was the mammoth steppe (unforested grassland), which
stretched across northern Eurasia and North America.
The woolly mammoth coexisted with early humans, who used its bones and tusks for making art,
tools, and dwellings (homes), and the species was also hunted for food, as one mammoth had
enough meet to feel a whole colony of early humans, thus it was an efficient diet for the early
humans. It disappeared from its mainland range at the end of the Pleistocene 10,000 years ago,
most likely through climate change; warming of the climate as they were adapted for cold climates
and may not have been able to survive higher temperatures, and thus resulting in shrinkage of its
habitat, hunting by humans, or a combination of the two. Isolated populations survived on St. Paul
Island until 6,400 years ago and Wrangel Island until 3,300 years ago.
INTRO
Recently since the invention and development of cloning it has made it possible that the woolly
mammoth can be brought back to life; de-extinction. This can be proposedly done by two methods.
The first is through the use of gene splicing, this is when the whole genome is studied and bits and
pieces of it is put into its close relative the Asian Elephant thus producing a hybrid with woolly
mammoth characteristics and traits, then cloning is used to produce a lot of individuals at once to
bring back a large population of the mammal. The second method which is less likely to occur is
where scientists will use the somatic cells of the naturally preserved (frozen) woolly mammoths to
create replicas of the individual that the cell came from. There are many biological implications
associated with cloning the mammoth back to existence and all must be taken into consideration
before the cloning of the mammal/

GENE SPLICING METHOD

The first method in which we can bring the woolly mammoth back to existence is gene splicing using
a DNA editing tool named CRISPR. Gene splicing involves cutting out a specific section of the DNA
in a gene which results in a specific trait being shown in the individual and adding new DNA in its
place. The process is entirely chemical with restriction enzymes used as chemical 'scissors'.
Depending on the type of restriction enzyme used, different parts of the genetic code can be
targeted. A specific restriction enzyme will split apart a specific strand of DNA leaving behind a gap
in the genetic code. New DNA can then be added in this gap.
When a new strand of DNA is added, it takes the place of the binds to the ends of the DNA strands
that were originally cut. Another enzyme called ligase is used in the repair process. Once the new
DNA is in place, the function of the gene will change and this produces a different phenotype or
characteristic expressed in the individual.

Geneticists have studied DNA from mammoths which were preserved in Arctic permafrost
(a thick subsurface layer of soil that remains below freezing point throughout the year,
occurring chiefly in polar regions) looking for genes which separated the mammoths from
elephants, such as hairiness and ear size. The researchers found that mammoth and elephant
DNA were 99.4% identical. This is actually similar to what has been found so far between humans
and Neanderthals.
Scientists at Harvard University then replicated the genes and spliced them into the genetic code of
an elephant where they functioned normally. It is the first time that mammoth genes have been alive
for more than 3,300 years - although so far it has only been done in the lab.
The goal for this method is to read out the phenotypes,- George Church at Harvard University
(person in charge for de-extinction of the mammal). To be able to accomplish this, the team needs to
decipher how to remove the flat hybrid cells (a gamete of an animal that is produced from two
animals of different kinds) from a petri dish (photo shown below) and arranging them carefully into
becoming specialized tissues- such as blood cells or liver organoids, then test to see if they behave
properly. For example a question that may arise is, do the mammoth hair genes lead to hair that's
the right colour, length, and woolliness?

Once the tests are complete, the next step is hopefully to turn the elephant/mammoth skin cells into
hybrid embryos that can be grown using the somatic cell cloning method (explained bellow),
implanting the hybrids into the wombs of female elephants is unfavourable to animal rights activists
as well as geneticists. Its going to be more humane and easier if we can set up hundreds of
embryos in an incubator and run tests, says Church.

If they can get the hybrid creatures to survive, the project's first goal will be to engineer an elephant
that can survive in cold temperatures. The team thinks that expanding the elephants range into
colder climates could help keep it away from humans and the conflicts that are threatening to make
Asian and African elephants extinct. Later, after the engineered elephants gain a foothold, Church
says the team will try to revive the mammoths by integrating higher amounts of mammoth DNA into
the hybrids.

Of course, it's possible the mammoth genome will never be completely

reconstructed, and the creatures will only remain elephant/mammoth
hybrids. But if it looks like a mammoth and fulfils the same ecological
functions as a mammoth, is it a mammoth? What even is a mammoth,
anyway?

Whole body organism cloning

The second method of cloning the mammoth back to existence is the whole body organism cloning.
This is the normal method of cloning just slightly tweaked to suit frozen species. This was tested on
a rat that was frozen for 16 years, and even though a 16 year old frozen rat is different to a 16,000
year old frozen mammoth it is a useful guideline that scientists are going by.
The process of somatic cell nuclear transplant (SCNT) involves two different cells. The first being a
female gamete, known as the ovum (egg/oocyte). The second being a somatic cell, referring to the
cells of the human body. Skin cells, fat cells, and liver cells are only a few examples. The most
commonly used are skin cells.

The nucleus of the donor egg cell is removed and discarded, leaving it 'deprogrammed.' The nucleus
of the somatic cell is also removed but is kept; the enucleated somatic cell is discarded. The
remaining somatic cell nucleus and an enucleated (cell without its nucleus) egg cell. These are then
fused by injecting the somatic nucleus into the 'empty' ovum. After being inserted into the egg, the
somatic cell nucleus is reprogrammed by its host egg cell. The ovum, now containing the somatic
cell's nucleus, is stimulated with a shock and this will cause the cell to begin to divide. The egg is
now viable and capable of producing an adult organism containing all the necessary genetic
information from just one parent. Development will result normally and after many mitotic divisions,
this single cell forms a blastocyst (an early stage embryo with about 100 cells) with an identical
genome to the original organism (i.e. a clone).

The problem with a frozen animal cell is that it is dead and ice crystals have damaged it. Up until the
study of the 16 year old frozen rat, it has not been possible to fuse a once frozen dead cell with an
enucleated egg.
Researchers of the frozen mouse got around the problem of frozen cells. Instead of fusing two cells,
they took nuclei out of the frozen cells and injected them directly into an enucleated egg. The
researchers established that a frozen brain has more intact nuclei than other frozen tissues. This is
due to the fact that the brain contains glucose which makes the freezing process gentler.

What they did was to add a nucleus from the long frozen mouse to an enucleated mouse egg and let
it grow for a while in a Petri dish. Then, instead of putting this growing embryo into a surrogate
mouse mother, the researchers isolated some embryonic stem (ES) cells from it. They then fused
one of these ES cells to another enucleated mouse egg. When the researchers implanted this
embryo into a surrogate mouse, they managed to get a clone. In fact, they got a total of four clones.
This means that scientists can clone mice that have been frozen for 16 years. Now they can try to
clone a mammoth that has been frozen for 16,000 using a similar method. It will obviously have
different challenges and since there are no surrogate mothers around they will either use an
incubator or they will require the uterus and enucleated egg cells of the mammoths common relative
the Asian elephant. The principal of the process will be the same also. This similarity makes it much
more likely that a mammoth nucleus will be tolerated by an elephant's egg. So it just might be
possible to clone a mammoth.

IMPLICATIONS
ECOSYSTEMS
The most significant implication for de-extinction of the woolly mammoth is that impact on the
ecosystem.
The woolly mammoths niche was similar to the grassy steppes of modern Russia, but the flora was
more diverse, abundant, and grew faster. Grasses, sedges, shrubs, and herbaceous plants were
present, and scattered trees were mainly found in southern regions. This habitat was not dominated
by ice and snow, as is popularly believed, since these regions are thought to have been highpressure areas at the time. This habitat will not be difficult to replicate in order to sustain the reexistence of the mammoth as we have technology to do this although, it will not be similar diet as
the types of flora do not grow there anymore due to the change in climate so there will need to be a
way to artificially feed them such as give them artificial nutrients such as how domesticated dogs eat
dog food.
The mammoths diet consisted of plant food, mainly grass and sedges, which were supplemented
with herbaceous plants, flowering plants, shrubs, mosses, and tree matter. Woolly mammoths
needed a varied diet to support their growth, like modern elephants. An adult of six tonnes would
need to eat 180 kg (397 lb) daily, and may have foraged as long as twenty hours every day. Since
the woolly mammoths diet is able to be replicated today there is no problem, it can still exist as it can
find a food source. But if they are released into the wild, they could possibly out compete the other
animals which eat the grass and plants in that certain niche, this could be significant as the animals
that were at balance are now not as they have another variable in the mix, causing them to not get
as much food and thus possibly their death and since they die they may not have a chance to
reproduce and pass on their genes and so it can lead to the extinction of the species of population
that the woolly mammoth is co-existing with.

The big disadvantage with cloning of the mammoth is that they will need to be managed and
completely dependent on humans for survival, as their natural ecosystem is no longer available or
the same to occupy. This means all their food intake and environmental conditions must be
completely controlled by humans in order for them to survive. They cannot be released into the wild
as there is no wild areas that can support the mammoths, also since they will have no parents
available to teach the animals how to behave the will need to be raised around Asian Elephants
since they have similar behavioural traits, or they will need to be raised around humans which can
teach them how to behave. If they are to be raised by Asian elephants mammoths may out-compete
the already dangerously close to extinction Asian elephants, if there is an added elephantidae
species it may out compete the existing ones as it is bigger and requires a larger diet. Research
suggests it may have foraged as long as twenty hours every day to maintain its dietary requirements
which will cause the mammoths to eat most of the food in the environment/niche, if the Asian
elephant and mammoth co-existed it would mean the mammoth would take up all the elephants food
source and thus causing the Asian elephants to be under nourished and this will lead to their death
as Asian elephants are smaller which means less food intake required which results in a smaller
time foraging. This will cause the mammoths to out compete the Asian elephants and can result in
the extinction of the existing species.

Another thing that could be devastating is that there may be specific pathogens and diseases that
these animals would carry that could kill us and thus possibly cause the extinction of the human
race. Additionally an impact to the ecosystem and to the human population is that a large population
of mammoths will cause higher levels of carbon dioxide emissions in the air due to added
respiration, carbon dioxide is a greenhouse gas and so will contribute to the greenhouse gas effect,
this means there will be more heat being trapped in the atmosphere as it is not able to get out as
easily thus can add to global warming theory which is a huge ecological devastation at a global
scale. The mammoth excretion may cause harmful chemical gases to be released into atmosphere
that could damage the ecosystem, for example cattle (cows) release methane gas as a part of their
normal digestive process and since methane gas is a greenhouse gas it can add to the global
warming problem.

-Will new species out-compete existing member of the community, and/or disrupt food chains/or
cause local extinctions?
-Will the cloned species be out-competed by existing species? Will food supplies still be in existence
and sufficient for the cloned species to survive?

IMPACTS ON GENETIC DIVERSITY

Cloning produces individuals that are genetically identical (including sex). In the specific case of
mammoths, to clone a population back to existence there is a need to produce many clones, and
since clones are the same genetically there will be no genetic diversity. This creates significant
disadvantages to the survival of the population as a whole as they are less likely to survive an
environmental change, especially the introduction of a new pathogen/disease. This is due to the fact
there is no variety in the genes and so if one is not immune to the disease, pathogen or

environmental then all of them are not immune and so the population could face being wiped out (reextinction).
An example of a species that has a low genetic diversity is the cheetah, about 12,000 years ago; a
mass extinction event occurred that eliminated 75% of the worlds large mammal species.
Fortunately, a handful of cheetahs managed to survive this extreme extinction event and were able
to restore the worlds population of cheetahs.
This event caused an extreme reduction of the cheetahs genetic diversity, known as a
population bottleneck, resulting in the physical homogeneity of todays cheetahs. Poor sperm
quality, focal palatine erosion, susceptibility to the same infectious diseases, and kinked tails
characteristic of the majority of the worlds cheetahs are all consequences of the low genetic
diversity within the global cheetah population.
Suitable levels of genetic diversity are vital to a populations ability to adapt and overcome
environmental changes and unexpected disasters. Unsustainable human expansion and
consumption leads to more unstable environmental conditions, and when habitat is destroyed
and fragmented, the rate of inbreeding increases, which leads to even more reduction in genetic
diversity. The coupling of these factors increases the risk of environmental variability to the
worlds cheetah population. Therefore, ensuring that the low levels of genetic diversity in the
worlds cheetah population do not decrease further is vital to the survival of the cheetah.
Genetic variation is the raw material of evolution. Without genetic variation, a population will not be
able to evolve in response to changing ecological/environmental variables and, as a consequence,
may face an increased risk of extinction. For example, if a population is exposed to a new disease,
individuals will be selected for if they have genes for resistance to the disease if they exist in the
population. But if they do not exist, if the right genetic variation is not present, the population will not
evolve and could potentially be wiped out by the disease.
As an endangered species decreases, it loses genetic variation, and even if the species rebounds,
its level of genetic variation will not. Genetic variation will only slowly be restored through the
accumulation of mutations over many generations. For this reason, an endangered species with low
genetic variation may risk extinction long after its population size has recovered.
Evolutionary theory suggests that, for the long-term survival of a species like the woolly mammoth,
we need to conserve not just individual members of a species, but also a species ability to evolve in
the event of changing environments, which means saving individuals and genetic variation.
The risk of extinction or population decline because of low genetic variation is predicted by
evolutionary theory. A case study of the cheetah, which has famously low genetic variation, suggests
the kinds of dangers that are likely. When the domesticated cats at an Oregon breeding colony for
large cats were exposed to a potentially deadly virus, it swept through the cheetah population, killing
about 50% as a direct or indirect result of the virus, but none of the lions even developed symptoms.
To conclude, it is possible that the mammoths low genetic variation meant that none of them had the
right immune system gene variants to fend off the disease, thus increasing their chance of reextinction.
.

IMPACT ON THE HEALTH AND SURVIVAL OF THE INDIVIDUAL

One significant factor that makes the cloning of living organisms so inefficient or devastating is the
fact that telomere shortening causes many health issues such as rapid aging, tumours, cancers,
organ failures and premature death.
Inside the nucleus of a cell, our genes are arranged along twisted, double-stranded molecules of
DNA called chromosomes. At the ends of the chromosomes are stretches of DNA called telomeres,
which protect our genetic data, make it possible for cells to divide.
Each time a cell divides, the telomeres get shorter. When the telomeres in a somatic cell get too
short, the cell can no longer divide; it becomes inactive or "senescent" or it dies. This shortening
process is associated with aging, cancer, and a higher risk of death. This process is shown in the
photo below; it also shows the same process in gametes.
Over an organism's lifetime, the DNA within its cells deteriorates, and unrepaired damage causes
some diseases, including cancer. Scientists are disturbed at the fact that this accumulated damage
may be carried along during the cloning process to the clone produced from the parent. If so, it
would give the clone a "head start" on developing some diseases and cancer. This is a significant
implication as it may cause the woolly mammoth to be born with many problems such as organ
failure as it did in dolly the sheep the first individual to be cloned, telomere shortening caused
damage to her brain, liver and heart, she also developed tumours growing in her lungs due to
incorrect cell division. This process will cause the cloned individual to suffer many serious illnesses

and will result in an early death so it is only once these glitches are fixed that the scientists should
look ahead to cloning.

Health and Survival of Species

Another huge problem is that there is possible risk to the health of the species. Inbreeding is defined
as: breed from closely related people or animals, especially over many generations. According to the
DNA research mammoths were severely inbred by the time the Wrangel Island mammoth was born.
This can cause inbreeding depression. The inbreeding that occurred between the mammoths may
lower the populations ability to survive and reproduce when brought back to existence, a
phenomenon called inbreeding depression. For example, a population of 40 adders (snake) when
through inbreeding depression when farming activities in Sweden isolated them from other adder
populations. Higher proportions of stillborn and deformed offspring were born in the isolated
population than in the larger populations. When researchers introduced adders from other
populations (an example of outbreeding) the isolated population then recovered and produced a
higher proportion of viable/normal offspring.
Over time, natural selection removes deleterious alleles out of a populationwhen the dominant
deleterious alleles are expressed, they lower the carriers fitness, and fewer copies are selected for
and so less will end up in the gene pool and thus less will end up in the next generation. But
recessive deleterious alleles are hidden from natural selection by their dominant non-deleterious
correspondents. An individual carrying a single recessive deleterious allele will be healthy and can
easily pass the deleterious allele into the next generation. This can be significant as if two
mammoths breed which both have recessive deleterious alleles (due to the fact that their ancestors
were heavily inbreed) it can cause their offspring to have deformities and even stillborn babies, this
can result in the population of mammoths not being able to further grow and thus causing their reextinction.

The solution to the inbreeding depression will be difficult as there is no other population of
mammoths to breed the cloned population with, so a possible way may be to breed the mammoth
with the Asian elephant to create a hybrid, but the hybrid may not be fertile (due to uneven
chromosomes causing the cells not being able to split for meiosis: gamete production), like the mule
a cross between a horse and a donkey. Inbreeding depression may endanger the survival of the
species.

SELECTIVE BREEDING
Selective breeding (also called artificial selection) is the process by which humans breed other
animals and plants for particular traits. Typically, species that are selectively bred are domesticated,
and the breeding is normally done by a professional breeder.
Around seven thousand years ago, the first bananas were discovered. In the wild, bananas were not
easy to eat. They probably had to be peeled using sharp rocks and whoever was eating them would
have had to constantly spit out big seeds. However, not all the bananas were exactly the same as
each other. Just like humans, they had plenty of diversity; some bananas were thinner than the
others, some had different skin colours, some were especially seedy and some were sweeter than
the rest.
Humans didnt fail to notice this diversity in the banana trees. So what they did was to collect the
bananas with the most desirable qualities they could find, and breed them. They found bananas with
the smallest seeds, bananas with the thinnest skin, bananas that were longer than the others, and
used them to plant new banana trees. Just like with humans, bananas pass on traits to their
offspring. A seed from a sweet banana is more likely to produce a sweet banana, just as a tall parent
is more likely to produce a tall child.
Over generations of breeding, bananas with desirable traits were bred and encouraged to grow,
while bananas with undesirable traits (i.e. fatter, bitterer and thicker ones) were tossed back into the

wild, where their chances of survival were much lower. Consequently, an entirely new breed of
banana was produced. These were bananas that had over the centuries been selectively bred so
that only those best suited for human consumption were refined, spread and became widely
available to humans.
The process of growing numerous plants from one plant by non-natural methods is called artificial
propagation. A number of methods of artificial propagation of plants are used in agriculture (for
raising crops), and horticulture (cultivation of vegetables, fruits and flowers). The two common
methods for the artificial propagation of bananas are; Cuttings and Grafting.
A small part of a plant which is removed by making a cut with a sharp knife is called a 'cutting'. A
cutting may be a piece of stem, root or even a leaf. While making a cut, the cutter must make sure
that there are some buds on it. In this method, a cutting of the parent plant (say, of stem or shoot)
having some buds on it is taken and its lower part is buried in the moist soil. After a few days, the
cutting develops roots; shoot, and grows into a new plant. Cuttings are a means of asexual
reproduction in plants. The new plant formed from a cutting is exactly similar to the parent plant.
Many plants such as bananas can be grown by means of cuttings. For example, rose plants are
propagated (or reproduced) by means cuttings from stems (or shoots) as follows: A piece of stem (or
side shoot) having bud is cut from an; existing rose plant with the help of a knife. The lower part of
this Gutting is buried in moist soil. After a few days, the end of cutting buried in soil develops roots
and later on grows to become a new rose plant. An advantage of cuttings method is that by using
this method we can produce many new plants from just one plant quickly.

Grafting is a method in which the cut stems of two different plants (one with roots and other without
roots) are joined together in such a way that the two stems join and grow as a single plant. This new
plant will have the characteristics of both the original plants. Artificial vegetative propagation is
usually used for the propagation (or reproduction) of those plants which produce either very few
seeds or do not produce viable seeds. Some examples of such plants which are reproduced by
artificial vegetative propagation methods are: Banana, Pineapple, Orange, Grape, Rose, etc.