Molecular cloning[edit]

Molecular cloning refers to the process of making multiple molecules.

Cloning is commonly used to amplify DNA fragments containing
whole genes, but it can also be used to amplify any DNA sequence such
as promoters, non-coding sequences and randomly fragmented DNA. It
is used in a wide array of biological experiments and practical
applications ranging from genetic fingerprinting to large scale protein
production. Occasionally, the term cloning is misleadingly used to refer
to the identification of the chromosomal location of a gene associated
with a particular phenotype of interest, such as in positional cloning. In
practice, localization of the gene to a chromosome or genomic region
does not necessarily enable one to isolate or amplify the relevant
genomic sequence. To amplify any DNA sequence in a living organism,
that sequence must be linked to an origin of replication, which is a
sequence of DNA capable of directing the propagation of itself and any
linked sequence. However, a number of other features are needed, and
a variety of specialised cloning vectors (small piece of DNA into which a
foreign DNA fragment can be inserted) exist that allow protein
production, affinity tagging, single-stranded RNA or DNA production and
a host of other molecular biology tools.
Cloning of any DNA fragment essentially involves four steps[8]

1. fragmentation - breaking apart a strand of DNA

2. ligation – gluing together pieces of DNA in a desired sequence
3. transfection – inserting the newly formed pieces of DNA into cells
4. screening/selection – selecting out the cells that were successfully
transfected with the new DNA
Although these steps are invariable among cloning procedures a number
of alternative routes can be selected; these are summarized as a cloning
strategy.
Initially, the DNA of interest needs to be isolated to provide a DNA
segment of suitable size. Subsequently, a ligation procedure is used
where the amplified fragment is inserted into a vector (piece of DNA).
The vector (which is frequently circular) is linearised using restriction
enzymes, and incubated with the fragment of interest under appropriate
conditions with an enzyme called DNA ligase. Following ligation, the
vector with the insert of interest is transfected into cells. A number of
alternative techniques are available, such as chemical sensitisation of
cells, electroporation, optical injection and biolistics. Finally, the
transfected cells are cultured. As the aforementioned procedures are of
particularly low efficiency, there is a need to identify the cells that have
been successfully transfected with the vector construct containing the
desired insertion sequence in the required orientation. Modern cloning
vectors include selectable antibiotic resistance markers, which allow
only cells in which the vector has been transfected, to grow.
Additionally, the cloning vectors may contain colour selection markers,
which provide blue/white screening (alpha-factor complementation)
on X-gal medium. Nevertheless, these selection steps do not absolutely
guarantee that the DNA insert is present in the cells obtained. Further
investigation of the resulting colonies must be required to confirm that
cloning was successful. This may be accomplished by means of PCR,
restriction fragment analysis and/or DNA sequencing.
Cell cloning[edit]
Cloning unicellular organisms[edit]

Cloning cell-line colonies using cloning rings

Cloning a cell means to derive a population of cells from a single cell. In

the case of unicellular organisms such as bacteria and yeast, this process
is remarkably simple and essentially only requires the inoculation of the
appropriate medium. However, in the case of cell cultures from multi-
cellular organisms, cell cloning is an arduous task as these cells will not
readily grow in standard media.
A useful tissue culture technique used to clone distinct lineages of cell
lines involves the use of cloning rings (cylinders).[9] In this technique a
single-cell suspension of cells that have been exposed to
a mutagenic agent or drug used to drive selection is plated at high
dilution to create isolated colonies, each arising from a single and
potentially clonal distinct cell. At an early growth stage when colonies
consist of only a few cells, sterile polystyrene rings (cloning rings), which
have been dipped in grease, are placed over an individual colony and a
small amount of trypsin is added. Cloned cells are collected from inside
the ring and transferred to a new vessel for further growth.
Cloning stem cells[edit]

Somatic-cell nuclear transfer, popularly known as SCNT, can also be used

to create embryos for research or therapeutic purposes. The most likely
purpose for this is to produce embryos for use in stem cell research. This
process is also called "research cloning" or "therapeutic cloning". The
goal is not to create cloned human beings (called "reproductive
cloning"), but rather to harvest stem cells that can be used to study
human development and to potentially treat disease. While a clonal
human blastocyst has been created, stem cell lines are yet to be isolated
from a clonal source.[10]
Therapeutic cloning is achieved by creating embryonic stem cells in the
hopes of treating diseases such as diabetes and Alzheimer's. The process
begins by removing the nucleus (containing the DNA) from an egg cell
and inserting a nucleus from the adult cell to be cloned.[11] In the case
of someone with Alzheimer's disease, the nucleus from a skin cell of that
patient is placed into an empty egg. The reprogrammed cell begins to
develop into an embryo because the egg reacts with the transferred
nucleus. The embryo will become genetically identical to the patient.
[11] The embryo will then form a blastocyst which has the potential to
form/become any cell in the body.[12]
The reason why SCNT is used for cloning is because somatic cells can be
easily acquired and cultured in the lab. This process can either add or
delete specific genomes of farm animals. A key point to remember is
that cloning is achieved when the oocyte maintains its normal functions
and instead of using sperm and egg genomes to replicate, the donor's
somatic cell nucleus is inserted into the oocyte.[13] The oocyte will react
to the somatic cell nucleus, the same way it would to a sperm cell's
nucleus.[13]
The process of cloning a particular farm animal using SCNT is relatively
the same for all animals. The first step is to collect the somatic cells from
the animal that will be cloned. The somatic cells could be used
immediately or stored in the laboratory for later use.[13] The hardest
part of SCNT is removing maternal DNA from an oocyte at metaphase II.
Once this has been done, the somatic nucleus can be inserted into an
egg cytoplasm.[13] This creates a one-cell embryo. The grouped somatic
cell and egg cytoplasm are then introduced to an electrical current.
[13] This energy will hopefully allow the cloned embryo to begin
development. The successfully developed embryos are then placed in
surrogate recipients, such as a cow or sheep in the case of farm animals.
[13]
SCNT is seen as a good method for producing agriculture animals for
food consumption. It successfully cloned sheep, cattle, goats, and pigs.
Another benefit is SCNT is seen as a solution to clone endangered
species that are on the verge of going extinct.[13] However, stresses
placed on both the egg cell and the introduced nucleus can be
enormous, which led to a high loss in resulting cells in early research. For
example, the cloned sheep Dolly was born after 277 eggs were used for
SCNT, which created 29 viable embryos. Only three of these embryos
survived until birth, and only one survived to adulthood.[14] As the
procedure could not be automated, and had to be performed manually
under a microscope, SCNT was very resource intensive. The biochemistry
involved in reprogramming the differentiated somatic cell nucleus and
activating the recipient egg was also far from being well understood.
However, by 2014 researchers were reporting cloning success rates of
seven to eight out of ten[15] and in 2016, a Korean Company Sooam
Biotech was reported to be producing 500 cloned embryos per day.[16]
In SCNT, not all of the donor cell's genetic information is transferred, as
the donor cell's mitochondria that contain their own mitochondrial
DNA are left behind. The resulting hybrid cells retain those
mitochondrial structures which originally belonged to the egg. As a
consequence, clones such as Dolly that are born from SCNT are not
perfect copies of the donor of the nucleus.
Organism cloning[edit]
See also: Asexual reproduction, Cuttings (plants), and vegetative
reproduction
Organism cloning (also called reproductive cloning) refers to the
procedure of creating a new multicellular organism, genetically identical
to another. In essence this form of cloning is an asexual method of
reproduction, where fertilization or inter-gamete contact does not take
place. Asexual reproduction is a naturally occurring phenomenon in
many species, including most plants and some insects. Scientists have
made some major achievements with cloning, including the asexual
reproduction of sheep and cows. There is a lot of ethical debate over
whether or not cloning should be used. However, cloning, or asexual
propagation,[17] has been common practice in the horticultural world
for hundreds of years.
Horticultural[edit]

Propagating plants from cuttings, such as grape vines, is an ancient

form of cloning.

For the use of cloning in viticulture, see Propagation of grapevines.

The term clone is used in horticulture to refer to descendants of a single
plant which were produced by vegetative reproduction or apomixis.
Many horticultural plant cultivars are clones, having been derived from a
single individual, multiplied by some process other than sexual
reproduction.[18] As an example, some European cultivars of grapes
represent clones that have been propagated for over two millennia.
Other examples are potato and banana.[19]
Grafting can be regarded as cloning, since all the shoots and branches
coming from the graft are genetically a clone of a single individual, but
this particular kind of cloning has not come under ethical scrutiny and is
generally treated as an entirely different kind of operation.
Many trees, shrubs, vines, ferns and other herbaceous
perennials form clonal colonies naturally. Parts of an individual plant
may become detached by fragmentation and grow on to become
separate clonal individuals. A common example is in the vegetative
reproduction of moss and liverwort gametophyte clones by means
of gemmae. Some vascular plants e.g. dandelion and
certain viviparous grasses also form seeds asexually, termed apomixis,
resulting in clonal populations of genetically identical individuals.
Parthenogenesis[edit]
Clonal derivation exists in nature in some animal species and is referred
to as parthenogenesis (reproduction of an organism by itself without a
mate). This is an asexual form of reproduction that is only found in
females of some insects, crustaceans, nematodes,[20] fish (for example
the hammerhead shark[21]), Cape honeybees,[22] and lizards including
the Komodo dragon[21] and several whiptails. The growth and
development occurs without fertilization by a male. In plants,
parthenogenesis means the development of an embryo from an
unfertilized egg cell, and is a component process of apomixis. In species
that use the XY sex-determination system, the offspring will always be
female. An example is the little fire ant (Wasmannia auropunctata),
which is native to Central and South America but has spread throughout
many tropical environments.
Artificial cloning of organisms[edit]

Artificial cloning of organisms may also be called reproductive cloning.

First steps[edit]

Hans Spemann, a German embryologist was awarded a Nobel Prize in

Physiology or Medicine in 1935 for his discovery of the effect now
known as embryonic induction, exercised by various parts of the
embryo, that directs the development of groups of cells into particular
tissues and organs. In 1924 he and his student, Hilde Mangold, were the
first to perform somatic-cell nuclear transfer using amphibian embryos –
one of the first steps towards cloning.[23]
Methods[edit]

Reproductive cloning generally uses "somatic cell nuclear transfer"

(SCNT) to create animals that are genetically identical. This process
entails the transfer of a nucleus from a donor adult cell (somatic cell) to
an egg from which the nucleus has been removed, or to a cell from
a blastocyst from which the nucleus has been removed.[24] If the egg
begins to divide normally it is transferred into the uterus of the
surrogate mother. Such clones are not strictly identical since the somatic
cells may contain mutations in their nuclear DNA. Additionally,
the mitochondria in the cytoplasm also contains DNA and during SCNT
this mitochondrial DNA is wholly from the cytoplasmic donor's egg, thus
the mitochondrial genome is not the same as that of the nucleus donor
cell from which it was produced. This may have important implications
for cross-species nuclear transfer in which nuclear-mitochondrial
incompatibilities may lead to death.
Artificial embryo splitting or embryo twinning, a technique that creates
monozygotic twins from a single embryo, is not considered in the same
fashion as other methods of cloning. During that procedure, a
donor embryo is split in two distinct embryos, that can then be
transferred via embryo transfer. It is optimally performed at the 6- to 8-
cell stage, where it can be used as an expansion of IVF to increase the
number of available embryos.[25] If both embryos are successful, it gives
rise to monozygotic (identical) twins.
Dolly the sheep[edit]

Main article: Dolly (sheep)

The taxidermied body of Dolly the sheep

 Dolly clone

Dolly, a Finn-Dorset ewe, was the first mammal to have been

successfully cloned from an adult somatic cell. Dolly was formed by
taking a cell from the udder of her 6-year-old biological mother.
[26] Dolly's embryo was created by taking the cell and inserting it into a
sheep ovum. It took 435 attempts before an embryo was successful.
[27] The embryo was then placed inside a female sheep that went
through a normal pregnancy.[28] She was cloned at the Roslin
Institute in Scotland by British scientists Sir Ian Wilmut and Keith
Campbell and lived there from her birth in 1996 until her death in 2003
when she was six. She was born on 5 July 1996 but not announced to the
world until 22 February 1997.[29] Her stuffed remains were placed at
Edinburgh's Royal Museum, part of the National Museums of Scotland.
[30]
Dolly was publicly significant because the effort showed that genetic
material from a specific adult cell, designed to express only a distinct
subset of its genes, can be redesigned to grow an entirely new organism.
Before this demonstration, it had been shown by John Gurdon that
nuclei from differentiated cells could give rise to an entire organism after
transplantation into an enucleated egg.[31] However, this concept was
not yet demonstrated in a mammalian system.
The first mammalian cloning (resulting in Dolly) had a success rate of 29
embryos per 277 fertilized eggs, which produced three lambs at birth,
one of which lived. In a bovine experiment involving 70 cloned calves,
one-third of the calves died quite young. The first successfully cloned
horse, Prometea, took 814 attempts. Notably, although the first clones
were frogs, no adult cloned frog has yet been produced from a somatic
adult nucleus donor cell.[32]
There were early claims that Dolly had pathologies resembling
accelerated aging. Scientists speculated that Dolly's death in 2003 was
related to the shortening of telomeres, DNA-protein complexes that
protect the end of linear chromosomes. However, other researchers,
including Ian Wilmut who led the team that successfully cloned Dolly,
argue that Dolly's early death due to respiratory infection was unrelated
to problems with the cloning process. This idea that the nuclei have not
irreversibly aged was shown in 2013 to be true for mice.[33]
Dolly was named after performer Dolly Parton because the cells cloned
to make her were from a mammary gland cell, and Parton is known for
her ample cleavage.[34]
Species cloned and applications[edit]

Further information: List of animals that have been cloned

Further information: Commercial animal cloning
The modern cloning techniques involving nuclear transfer have been
successfully performed on several species. Notable experiments include:

 Tadpole: (1952) Robert Briggs and Thomas J. King had successfully

cloned northern leopard frogs: thirty-five complete embryos and
twenty-seven tadpoles from one-hundred and four successful nuclear
transfers.[35][36]
 Carp: (1963) In China, embryologist Tong Dizhou produced the
world's first cloned fish by inserting the DNA from a cell of a male
carp into an egg from a female carp. He published the findings in a
Chinese science journal.[37]
 Zebrafish: The first vertebrate cloned (1981) by George
Streisinger (Streisinger, George; Walker, C.; Dower, N.; Knauber, D.;
Singer, F. (1981), "Production of clones of homozygous diploid zebra
fish (Brachydanio rerio)", Nature, 291 (5813): 293–
296, Bibcode:1981Natur.291..293S, doi:10.1038/291293a0, PMID 724
8006, S2CID 4323945)
 Sheep: Marked the first mammal being cloned (1984) from early
embryonic cells by Steen Willadsen. Megan and Morag[38] cloned
from differentiated embryonic cells in June 1995 and Dolly from a
somatic cell in 1996.[39][37]
 Mice: (1986) A mouse was successfully cloned from an early
embryonic cell. Soviet scientists Chaylakhyan, Veprencev, Sviridova,
and Nikitin had the mouse "Masha" cloned. Research was published
in the magazine Biofizika volume ХХХII, issue 5 of 1987.[clarification
needed][40][41][needs update]
 Rhesus monkey: Tetra (January 2000) from embryo splitting and not
nuclear transfer. More akin to artificial formation of twins.[42][43]
 Pig: the first cloned pigs (March 2000).[44] By 2014, BGI in China was
producing 500 cloned pigs a year to test new medicines.[45]
 Gaur: (2001) was the first endangered species cloned.[46]
 Cattle:
 o Alpha and Beta (males, 2001) and (2005), Brazil[47]
o In 2023, Chinese scientists reported the cloning of
three supercows with a milk productivity "nearly 1.7 times the
amount of milk an average cow in the United States produced in
2021" and a plan for 1,000 of such super cows in the near-term.
According to a news report "[i]n many countries, including the
United States, farmers breed clones with conventional animals to
add desirable traits, such as high milk production or disease
resistance, into the gene pool".[clarification needed][when?][48]

 Cat: CopyCat "CC" (female, late 2001), Little Nicky, 2004, was the first
cat cloned for commercial reasons[49]
 Rat: Ralph, the first cloned rat (2003)[50]
 Mule: Idaho Gem, a john mule born 4 May 2003, was the first horse-
family clone.[51]
 Horse: Prometea, a Haflinger female born 28 May 2003, was the first
horse clone.[52]
 Dog:
o Snuppy, a male Afghan hound was the first cloned dog (2005).
[53] In 2017, the world's first gene-editing clone dog, Apple, was
created by Sinogene Biotechnology.[54] Sooam Biotech, South
Korea, was reported in 2015 to have cloned 700 dogs to date for
their owners, including two Yakutian Laika hunting dogs, which are
seriously endangered due to crossbreeding.[55]
o Cloning of super sniffer dogs was reported in 2011, four years
afterwards when the dogs started working.[56] Cloning of a
successful rescue dog was also reported in 2009[57] and of a
similar police dog in 2019.[58] Cancer-sniffing dogs have also been
cloned. A review concluded that "qualified elite working dogs can
be produced by cloning a working dog that exhibits both an
appropriate temperament and good health."[59]
 Wolf: Snuwolf and Snuwolffy, the first two cloned female wolves
(2005).[60]
 Water buffalo: Samrupa was the first cloned water buffalo. It was
born on 6 February 2009, at India's Karnal National Diary Research
Institute but died five days later due to lung infection.[61]
 Pyrenean ibex (2009) was the first extinct animal to be cloned back to
life; the clone lived for seven minutes before dying of lung defects.
[62][63]
 Camel: (2009) Injaz, was the first cloned camel.[64]
 Pashmina goat: (2012) Noori, is the first cloned pashmina goat.
Scientists at the faculty of veterinary sciences and animal husbandry
of Sher-e-Kashmir University of Agricultural Sciences and Technology
of Kashmir successfully cloned the first Pashmina goat (Noori) using
the advanced reproductive techniques under the leadership of Riaz
Ahmad Shah.[65]
 Goat: (2001) Scientists of Northwest A&F University successfully
cloned the first goat which use the adult female cell.[66]
 Gastric brooding frog: (2013) The gastric brooding
frog, Rheobatrachus silus, thought to have been extinct since 1983
was cloned in Australia, although the embryos died after a few days.
[67]
 Macaque monkey: (2017) First successful cloning of a primate species
using nuclear transfer, with the birth of two live clones named Zhong
Zhong and Hua Hua. Conducted in China in 2017, and reported in
January 2018.[68][69][70][71] In January 2019, scientists in China
reported the creation of five identical cloned gene-edited monkeys,
using the same cloning technique that was used with Zhong Zhong
and Hua Hua and Dolly the sheep, and the gene-editing Crispr-
Cas9 technique allegedly used by He Jiankui in creating the first ever
gene-modified human babies Lulu and Nana. The monkey clones
were made to study several medical diseases.[72][73]
 Black-footed ferret: (2020) A team of scientists cloned a female
named Willa, who died in the mid-1980s and left no living
descendants. Her clone, a female named Elizabeth Ann, was born on
10 December. Scientists hope that the contribution of this individual
will alleviate the effects of inbreeding and help black-footed ferrets
better cope with plague. Experts estimate that this female's genome
contains three times as much genetic diversity as any of the modern
black-footed ferrets.[74]
 First artificial parthenogenesis in mammals: (2022) Viable mice
offspring was born from unfertilized eggs via targeted DNA
methylation editing of seven imprinting control regions.[75]
Human cloning[edit]

Main article: Human cloning

Human cloning is the creation of a genetically identical copy of a human.
The term is generally used to refer to artificial human cloning, which is
the reproduction of human cells and tissues. It does not refer to the
natural conception and delivery of identical twins. The possibility of
human cloning has raised controversies. These ethical concerns have
prompted several nations to pass legislation regarding human cloning
and its legality. As of right now, scientists have no intention of trying to
clone people and they believe their results should spark a wider
discussion about the laws and regulations the world needs to regulate
cloning.[76]
Two commonly discussed types of theoretical human cloning
are therapeutic cloning and reproductive cloning. Therapeutic cloning
would involve cloning cells from a human for use in medicine and
transplants, and is an active area of research, but is not in medical
practice anywhere in the world, as of 2021. Two common methods of
therapeutic cloning that are being researched are somatic-cell nuclear
transfer and, more recently, pluripotent stem cell induction.
Reproductive cloning would involve making an entire cloned human,
instead of just specific cells or tissues.[77]
Ethical issues of cloning[edit]

Main article: Ethics of cloning

There are a variety of ethical positions regarding the possibilities of
cloning, especially human cloning. While many of these views are
religious in origin, the questions raised by cloning are faced by secular
perspectives as well. Perspectives on human cloning are theoretical, as
human therapeutic and reproductive cloning are not commercially used;
animals are currently cloned in laboratories and in livestock production.
Advocates support development of therapeutic cloning to generate
tissues and whole organs to treat patients who otherwise cannot obtain
transplants,[78] to avoid the need for immunosuppressive drugs,
[77] and to stave off the effects of aging.[79] Advocates for reproductive
cloning believe that parents who cannot otherwise procreate should
have access to the technology.[80]
Opponents of cloning have concerns that technology is not yet
developed enough to be safe[81] and that it could be prone to abuse
(leading to the generation of humans from whom organs and tissues
would be harvested),[82][83] as well as concerns about how cloned
individuals could integrate with families and with society at large.[84]
[85] Cloning humans could lead to serious violations of human rights.
[86]
Religious groups are divided, with some opposing the technology as
usurping "God's place" and, to the extent embryos are used, destroying
a human life; others support therapeutic cloning's potential life-saving
benefits.[87][88] There is at least one religion, Raëlism, in which cloning
plays a major role.[89][90][91]
Contemporary work on this topic is concerned with the ethics, adequate
regulation and issues of any cloning carried out by humans, not
potentially by extraterrestrials (including in the future), and largely also
not replication – also described as mind cloning[92][93][94][95] – of
potential whole brain emulations.
Cloning of animals is opposed by animal-groups due to the number of
cloned animals that suffer from malformations before they die, and
while food from cloned animals has been approved as safe by the US
FDA,[96][97] its use is opposed by groups concerned about food safety.
[98][99]
In practical terms, the inclusion of "licensing requirements for embryo
research projects and fertility clinics, restrictions on the commodification
of eggs and sperm, and measures to prevent proprietary interests from
monopolizing access to stem cell lines" in international cloning
regulations has been proposed, albeit e.g. effective oversight
mechanisms or cloning requirements have not been described.[100]
Cloning extinct and endangered species[edit]

Main article: De-extinction

Cloning, or more precisely, the reconstruction of functional DNA
from extinct species has, for decades, been a dream. Possible
implications of this were dramatized in the 1984 novel Carnosaur and
the 1990 novel Jurassic Park.[101][102] The best current cloning
techniques have an average success rate of 9.4 percent[103] (and as
high as 25 percent[33]) when working with familiar species such as mice,
[note 1] while cloning wild animals is usually less than 1 percent
successful.[106]
Conservation cloning[edit]

Several tissue banks have come into existence, including the "Frozen
zoo" at the San Diego Zoo, to store frozen tissue from the world's rarest
and most endangered species.[101][107][108][109] This is also referred
to as "Conservation cloning".[110][111]
Engineers have proposed a "lunar ark" in 2021 – storing millions of seed,
spore, sperm and egg samples from Earth's contemporary species in a
network of lava tubes on the Moon as a genetic backup.[112][113]
[114] Similar proposals have been made since at least 2008.[115] These
also include sending human customer DNA,[116] and a proposal for "a
lunar backup record of humanity" that includes genetic information
by Avi Loeb et al.[117]
Scientists at the University of Newcastle and University of New South
Wales announced in March 2013 that the very recently extinct gastric-
brooding frog would be the subject of a cloning attempt to resurrect the
species.[118]
Many such "De-extinction" projects are described in the Long Now
Foundation's Revive and Restore Project.[119]
De-extinction[edit]

One of the most anticipated targets for cloning was once the woolly
mammoth, but attempts to extract DNA from frozen mammoths have
been unsuccessful, though a joint Russo-Japanese team is currently
working toward this goal.[when?] In January 2011, it was reported by
Yomiuri Shimbun that a team of scientists headed by Akira Iritani of
Kyoto University had built upon research by Dr. Wakayama, saying that
they will extract DNA from a mammoth carcass that had been preserved
in a Russian laboratory and insert it into the egg cells of an Asian
elephant in hopes of producing a mammoth embryo. The researchers
said they hoped to produce a baby mammoth within six years.[120]
[121] It was noted, however that the result, if possible, would be an
elephant-mammoth hybrid rather than a true mammoth.[122] Another
problem is the survival of the reconstructed mammoth: ruminants rely
on a symbiosis with specific microbiota in their stomachs for digestion.
[122]
In 2022, scientists showed major limitations and the scale of challenge of
genetic-editing-based de-extinction, suggesting resources spent on more
comprehensive de-extinction projects such as of the woolly
mammoth may currently not be well allocated and substantially limited.
Their analyses "show that even when the extremely high-quality Norway
brown rat (R. norvegicus) is used as a reference, nearly 5% of the
genome sequence is unrecoverable, with 1,661 genes recovered at
lower than 90% completeness, and 26 completely absent", complicated
further by that "distribution of regions affected is not random, but for
example, if 90% completeness is used as the cutoff, genes related to
immune response and olfaction are excessively affected" due to which
"a reconstructed Christmas Island rat would lack attributes likely critical
to surviving in its natural or natural-like environment".[123]
In a 2021 online session of the Russian Geographical Society, Russia's
defense minister Sergei Shoigu mentioned using the DNA of 3,000-year-
old Scythian warriors to potentially bring them back to life. The idea was
described as absurd at least at this point in news reports and it was
noted that Scythians likely weren't skilled warriors by default.[124][125]
[126]
The idea of cloning Neanderthals or bringing them back to life in general
is controversial but some scientists have stated that it may be possible in
the future and have outlined several issues or problems with such as
well as broad rationales for doing so.[127][128][129][130][131][132]
Unsuccessful attempts[edit]

In 2001, a cow named Bessie gave birth to a cloned Asian gaur, an

endangered species, but the calf died after two days. In 2003,
a banteng was successfully cloned, followed by three African
wildcats from a thawed frozen embryo. These successes provided hope
that similar techniques (using surrogate mothers of another species)
might be used to clone extinct species. Anticipating this possibility, tissue
samples from the last bucardo (Pyrenean ibex) were frozen in liquid
nitrogen immediately after it died in 2000. Researchers are also
considering cloning endangered species such as the Giant
panda and Cheetah.[133][134][135][136]
In 2002, geneticists at the Australian Museum announced that they had
replicated DNA of the thylacine (Tasmanian tiger), at the time extinct for
about 65 years, using polymerase chain reaction.[137] However, on 15
February 2005 the museum announced that it was stopping the project
after tests showed the specimens' DNA had been too badly degraded by
the (ethanol) preservative. On 15 May 2005 it was announced that the
thylacine project would be revived, with new participation from
researchers in New South Wales and Victoria.[138]
In 2003, for the first time, an extinct animal, the Pyrenean ibex
mentioned above was cloned, at the Centre of Food Technology and
Research of Aragon, using the preserved frozen cell nucleus of the skin
samples from 2001 and domestic goat egg-cells. The ibex died shortly
after birth due to physical defects in its lungs.[139]
Lifespan[edit]
After an eight-year project involving the use of a pioneering cloning
technique, Japanese researchers created 25 generations of healthy
cloned mice with normal lifespans, demonstrating that clones are not
intrinsically shorter-lived than naturally born animals.[33][140] Other
sources have noted that the offspring of clones tend to be healthier than
the original clones and indistinguishable from animals produced
naturally.[141]
Some posited that Dolly the sheep may have aged more quickly than
naturally born animals, as she died relatively early for a sheep at the age
of six. Ultimately, her death was attributed to a respiratory illness, and
the "advanced aging" theory is disputed.[142][dubious – discuss]
A detailed study released in 2016 and less detailed studies by others
suggest that once cloned animals get past the first month or two of life
they are generally healthy. However, early pregnancy loss and neonatal
losses are still greater with cloning than natural conception or assisted
reproduction (IVF). Current research is attempting to overcome these
problems.[34]
In popular culture[edit]

Sontarans in Doctor Who are a cloned warrior race.

Discussion of cloning in the popular media often presents the subject

negatively. In an article in the 8 November 1993 article of Time, cloning
was portrayed in a negative way, modifying Michelangelo's Creation of
Adam to depict Adam with five identical hands.[143] Newsweek's 10
March 1997 issue also critiqued the ethics of human cloning, and
included a graphic depicting identical babies in beakers.[144]
The concept of cloning, particularly human cloning, has featured a wide
variety of science fiction works. An early fictional depiction of cloning
is Bokanovsky's Process which features in Aldous Huxley's 1931
dystopian novel Brave New World. The process is applied to fertilized
human eggs in vitro, causing them to split into identical genetic copies of
the original.[145][146] Following renewed interest in cloning in the
1950s, the subject was explored further in works such as Poul
Anderson's 1953 story UN-Man, which describes a technology called
"exogenesis", and Gordon Rattray Taylor's book The Biological Time
Bomb, which popularised the term "cloning" in 1963.[147]
Cloning is a recurring theme in a number of contemporary science fiction
films, ranging from action films such as Anna to the Infinite Power, The
Boys from Brazil, Jurassic Park (1993), Alien Resurrection (1997), The 6th
Day (2000), Resident Evil (2002), Star Wars: Episode II – Attack of the
Clones (2002), The Island (2005) and Moon (2009) to comedies such
as Woody Allen's 1973 film Sleeper.[148]
The process of cloning is represented variously in fiction. Many works
depict the artificial creation of humans by a method of growing cells
from a tissue or DNA sample; the replication may be instantaneous, or
take place through slow growth of human embryos in artificial wombs.
In the long-running British television series Doctor Who, the Fourth
Doctor and his companion Leela were cloned in a matter of seconds
from DNA samples ("The Invisible Enemy", 1977) and then – in an
apparent homage to the 1966 film Fantastic Voyage – shrunk to
microscopic size to enter the Doctor's body to combat an alien virus. The
clones in this story are short-lived, and can only survive a matter of
minutes before they expire.[149] Science fiction films such as The
Matrix and Star Wars: Episode II – Attack of the Clones have featured
scenes of human foetuses being cultured on an industrial scale in
mechanical tanks.[150]
Cloning humans from body parts is also a common theme in science
fiction. Cloning features strongly among the science fiction conventions
parodied in Woody Allen's Sleeper, the plot of which centres around an
attempt to clone an assassinated dictator from his disembodied nose.
[151] In the 2008 Doctor Who story "Journey's End", a duplicate version
of the Tenth Doctor spontaneously grows from his severed hand, which
had been cut off in a sword fight during an earlier episode.[152]
After the death of her beloved 14-year-old Coton de Tulear named
Samantha in late 2017, Barbra Streisand announced that she had cloned
the dog, and was now "waiting for [the two cloned pups] to get older so
[she] can see if they have [Samantha's] brown eyes and her
seriousness".[153] The operation cost $50,000 through the pet cloning
company ViaGen.[154]
Cloning and identity[edit]

Science fiction has used cloning, most commonly and specifically human
cloning, to raise the controversial questions of identity.[155][156] A
Number is a 2002 play by English playwright Caryl Churchill which
addresses the subject of human cloning and identity, especially nature
and nurture. The story, set in the near future, is structured around the
conflict between a father (Salter) and his sons (Bernard 1, Bernard 2, and
Michael Black) – two of whom are clones of the first one. A Number was
adapted by Caryl Churchill for television, in a co-production between
the BBC and HBO Films.[157]
In 2012, a Japanese television series named "Bunshin" was created. The
story's main character, Mariko, is a woman studying child welfare in
Hokkaido. She grew up always doubtful about the love from her mother,
who looked nothing like her and who died nine years before. One day,
she finds some of her mother's belongings at a relative's house, and
heads to Tokyo to seek out the truth behind her birth. She later
discovered that she was a clone.[158]
In the 2013 television series Orphan Black, cloning is used as a scientific
study on the behavioral adaptation of the clones.[159] In a similar vein,
the book The Double by Nobel Prize winner José Saramago explores the
emotional experience of a man who discovers that he is a clone.[160]
Cloning as resurrection[edit]

Cloning has been used in fiction as a way of recreating historical figures.

In the 1976 Ira Levin novel The Boys from Brazil and its 1978 film
adaptation, Josef Mengele uses cloning to create copies of Adolf Hitler.
[161]
In Michael Crichton's 1990 novel Jurassic Park, which spawned a series
of Jurassic Park feature films, the bioengineering company InGen
develops a technique to resurrect extinct species of dinosaurs by
creating cloned creatures using DNA extracted from fossils. The cloned
dinosaurs are used to populate the Jurassic Park wildlife park for the
entertainment of visitors. The scheme goes disastrously wrong when the
dinosaurs escape their enclosures. Despite being selectively cloned as
females to prevent them from breeding, the dinosaurs develop the
ability to reproduce through parthenogenesis.[162]
Cloning for warfare[edit]

The use of cloning for military purposes has also been explored in
several fictional works. In Doctor Who, an alien race of armour-clad,
warlike beings called Sontarans was introduced in the 1973 serial "The
Time Warrior". Sontarans are depicted as squat, bald creatures who
have been genetically engineered for combat. Their weak spot is a
"probic vent", a small socket at the back of their neck which is associated
with the cloning process.[163] The concept of cloned soldiers being bred
for combat was revisited in "The Doctor's Daughter" (2008), when the
Doctor's DNA is used to create a female warrior called Jenny.[164]
The 1977 film Star Wars was set against the backdrop of a historical
conflict called the Clone Wars. The events of this war were not fully
explored until the prequel films Attack of the Clones (2002) and Revenge
of the Sith (2005), which depict a space war waged by a massive army of
heavily armoured clone troopers that leads to the foundation of
the Galactic Empire. Cloned soldiers are "manufactured" on an industrial
scale, genetically conditioned for obedience and combat effectiveness. It
is also revealed that the popular character Boba Fett originated as a
clone of Jango Fett, a mercenary who served as the genetic template for
the clone troopers.[165][166]
Cloning for exploitation[edit]

A recurring sub-theme of cloning fiction is the use of clones as a supply

of organs for transplantation. The 2005 Kazuo Ishiguro novel Never Let
Me Go and the 2010 film adaption[167] are set in an alternate history in
which cloned humans are created for the sole purpose of providing
organ donations to naturally born humans, despite the fact that they are
fully sentient and self-aware. The 2005 film The Island[168] revolves
around a similar plot, with the exception that the clones are unaware of
the reason for their existence.
The exploitation of human clones for dangerous and undesirable work
was examined in the 2009 British science fiction film Moon.[169] In the
futuristic novel Cloud Atlas and subsequent film, one of the story lines
focuses on a genetically engineered fabricant clone named Sonmi~451,
one of millions raised in an artificial "wombtank", destined to serve from
birth. She is one of thousands created for manual and emotional labor;
Sonmi herself works as a server in a restaurant. She later discovers that
the sole source of food for clones, called 'Soap', is manufactured from
the clones themselves.[170]
Will cloning bring the woolly mammoth back to life?
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In biomedical research, cloning is broadly defined to mean the duplication of

any kind of biological material for scientific study, such as a piece of DNA or
an individual cell. For example, segments of DNA are replicated exponentially
by a process known as polymerase chain reaction, or PCR, a technique that is
used widely in basic biological research. The type of cloning that is the focus of
much ethical controversy involves the generation of cloned embryos,
particularly those of humans, which are genetically identical to the organisms
from which they are derived, and the subsequent use of these embryos for
research, therapeutic, or reproductive purposes.

Early cloning experiments

Reproductive cloning was originally carried out by artificial “twinning,”
or embryo splitting, which was first performed on a salamander embryo in the
early 1900s by German embryologist Hans Spemann. Later, Spemann, who
was awarded the Nobel Prize for Physiology or Medicine (1935) for his
research on embryonic development, theorized about another cloning
procedure known as nuclear transfer. This procedure was performed in 1952
by American scientists Robert W. Briggs and Thomas J. King, who used DNA
from embryonic cells of the frog Rana pipiens to generate cloned tadpoles. In
1958 British biologist John Bertrand Gurdon successfully carried out nuclear
transfer using DNA from adult intestinal cells of African clawed frogs
(Xenopus laevis). Gurdon was awarded a share of the 2012 Nobel Prize in
Physiology or Medicine for this breakthrough.

Discover the cloning of Dolly the sheep by using somatic cell nuclear transfer
(SCNT)
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Advancements in the field of molecular biology led to the development of

techniques that allowed scientists to manipulate cells and to detect chemical
markers that signal changes within cells. With the advent of recombinant DNA
technology in the 1970s, it became possible for scientists to create transgenic
clones—clones with genomes containing pieces of DNA from other organisms.
Beginning in the 1980s mammals such as sheep were cloned from early and
partially differentiated embryonic cells. In 1996 British developmental
biologist Ian Wilmut generated a cloned sheep, named Dolly, by means of
nuclear transfer involving an enucleated embryo and a differentiated cell
nucleus. This technique, which was later refined and became known
as somatic cell nuclear transfer (SCNT), represented an extraordinary advance
in the science of cloning, because it resulted in the creation of a genetically
identical clone of an already grown sheep. It also indicated that it was possible
for the DNA in differentiated somatic (body) cells to revert to an
undifferentiated embryonic stage, thereby reestablishing pluripotency—the
potential of an embryonic cell to grow into any one of the numerous different
types of mature body cells that make up a complete organism. The realization
that the DNA of somatic cells could be reprogrammed to a pluripotent state
significantly impacted research into therapeutic cloning and the development
of stem cell therapies.

CC, the first cloned cat

Soon after the generation of Dolly, a number of other animals were cloned by
SCNT, including pigs, goats, rats, mice, dogs, horses, and mules. Despite those
successes, the birth of a viable SCNT primate clone would not come to fruition
until 2018, and scientists used other cloning processes in the meantime. In
2001 a team of scientists cloned a rhesus monkey through a process
called embryonic cell nuclear transfer, which is similar to SCNT except that it
uses DNA from an undifferentiated embryo. In 2007 macaque monkey
embryos were cloned by SCNT, but those clones lived only to
the blastocyst stage of embryonic development. It was more than 10 years
later, after improvements to SCNT had been made, that scientists announced
the live birth of two clones of the crab-eating macaque (Macaca fascicularis),
the first primate clones using the SCNT process. (SCNT has been carried out
with very limited success in humans, in part because of problems with human
egg cells resulting from the mother’s age and environmental factors.)

Therapeutic cloning
Therapeutic cloning is intended to use cloned embryos for the purpose of
extracting stem cells from them, without ever implanting the embryos in a
womb. Therapeutic cloning enables the cultivation of stem cells that are
genetically identical to a patient. The stem cells could be stimulated
to differentiate into any of the more than 200 cell types in the human body.
The differentiated cells then could be transplanted into the patient to replace
diseased or damaged cells without the risk of rejection by the immune system.
These cells could be used to treat a variety of conditions, including Alzheimer
disease, Parkinson disease, diabetes mellitus, stroke, and spinal cord injury.
In addition, stem cells could be used for in vitro (laboratory) studies of normal
and abnormal embryo development or for testing drugs to see if they are toxic
or cause birth defects.
Although stem cells have been derived from the cloned embryos of animals
such as mice, the generation of stem cells from cloned primate embryos has
proved exceptionally difficult. For example, in 2007 stem cells successfully
derived from cloned macaque embryos were able to differentiate into mature
heart cells and brain neurons. However, the experiment started with 304 egg
cells and resulted in the development of only two lines of stem cells, one of
which had an abnormal Y chromosome. Likewise, the production of stem cells
from human embryos has been fraught with the challenge of maintaining
embryo viability. In 2001 scientists at Advanced Cell Technology, a research
company in Massachusetts, successfully transferred DNA from human
cumulus cells, which are cells that cling to and nourish human eggs, into eight
enucleated eggs. Of these eight eggs, three developed into early-stage embryos
(containing four to six cells); however, the embryos survived only long enough
to divide once or twice. In 2004 South Korean researcher Hwang Woo
Suk claimed to have cloned human embryos using SCNT and to have extracted
stem cells from the embryos. However, this later proved to be a fraud; Hwang
had fabricated evidence and had actually carried out the process
of parthenogenesis, in which an unfertilized egg begins to divide with only half
a genome. The following year a team of researchers from the University
of Newcastle upon Tyne was able to grow a cloned human embryo to the 100-
cell blastocyst stage using DNA from embryonic stem cells, though they did
not generate a line of stem cells from the blastocyst. Scientists have since
successfully derived embryonic stem cells from SCNT human embryos.

Progress in research on therapeutic cloning in humans has been slow relative

to the advances made in reproductive cloning in animals. This is primarily
because of the technical challenges and ethical controversy arising from the
procuring of human eggs solely for research purposes. In addition, the
development of induced pluripotent stem cells, which are derived from
somatic cells that have been reprogrammed to an embryonic state through the
introduction of specific genetic factors into the cell nuclei, has challenged the
use of cloning methods and of human eggs.

Ethical controversy
Human reproductive cloning remains universally condemned, primarily for
the psychological, social, and physiological risks associated with cloning. A
cloned embryo intended for implantation into a womb requires thorough
molecular testing to fully determine whether an embryo is healthy and
whether the cloning process is complete. In addition, as demonstrated by 100
failed attempts to generate a cloned macaque in 2007, a viable pregnancy is
not guaranteed. Because the risks associated with reproductive cloning in
humans introduce a very high likelihood of loss of life, the process is
considered unethical. There are other philosophical issues that also have been
raised concerning the nature of reproduction and human identity that
reproductive cloning might violate. Concerns about eugenics, the once popular
notion that the human species could be improved through the selection of
individuals possessing desired traits, also have surfaced, since cloning could
be used to breed “better” humans, thus violating principles of human dignity,
freedom, and equality.
Learn about cloning for conservation and how scientists cloned the world's first
endangered species
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There also exists controversy over the ethics of therapeutic and research
cloning. Some individuals and groups have an objection to therapeutic
cloning, because it is considered the manufacture and destruction of a human
life, even though that life has not developed past the embryonic stage. Those
who are opposed to therapeutic cloning believe that the technique supports
and encourages acceptance of the idea that human life can be created and
expended for any purpose. However, those who support therapeutic cloning
believe that there is a moral imperative to heal the sick and to seek greater
scientific knowledge. Many of these supporters believe that therapeutic and
research cloning should be not only allowed but also publicly funded, similar
to other types of disease and therapeutics research. Most supporters also
argue that the embryo demands special moral consideration, requiring
regulation and oversight by funding agencies. In addition, it is important to
many philosophers and policy makers that women and couples not be
exploited for the purpose of obtaining their embryos or eggs.

There are laws and international conventions that attempt to uphold

certain ethical principles and regulations concerning cloning. In 2005
the United Nations passed a nonbinding Declaration on Human Cloning that
calls upon member states “to adopt all measures necessary to prohibit all
forms of human cloning inasmuch as they are incompatible with human
dignity and the protection of human life.” This does provide leeway for
member countries to pursue therapeutic cloning. The United Kingdom,
through its Human Fertilisation and Embryology Authority, issues licenses for
creating human embryonic stem cells through nuclear transfer. These licenses
ensure that human embryos are cloned for legitimate therapeutic and research
purposes aimed at obtaining scientific knowledge about disease and human
development. The licenses require the destruction of embryos by the 14th day
of development, since this is when embryos begin to develop the primitive
streak, the first indicator of an organism’s nervous system. The United
States federal government has not passed any laws regarding human cloning
due to disagreement within the legislative branch about whether to ban all
cloning or to ban only reproductive cloning. The Dickey-Wicker amendment,
attached to U.S. appropriations bills since 1995, has prevented the use of
federal dollars to fund the harm or destruction of human embryos for
research. It is presumed that nuclear transfer and any other form of cloning is
subject to this restriction.

Michael Rugnetta
tissue engineering
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Chapter 4.

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HOW DOES REPRODUCTIVE CLONING DIFFER FROM STEM CELL

RESEARCH?

The recent and current work on stem cells that is briefly summarized below and
discussed more fully in a recent report from the National Academies entitled Stem
Cells and the Future of Regenerative Medicine [ 11] is not directly related to human
reproductive cloning. However, the use of a common initial step—called either
nuclear transplantation or somatic cell nuclear transfer (SCNT)—has led Congress to
consider bills that ban not only human reproductive cloning but also certain areas of
stem cell research. Stem cells are cells that have the ability to divide repeatedly and
give rise to both specialized cells and more stem cells. Some, such as some blood and
brain stem cells, can be derived directly from adults [ 12- 19] and others can be
obtained from preimplantation embryos. Stem cells derived from embryos are called
embryonic stem cells (ES cells). The above-mentioned report from the National
Academies provides a detailed account of the current state of stem cell research [ 11].

ES cells are also called pluripotent stem cells because their progeny include all cell
types that can be found in a postimplantation embryo, a fetus, and a fully developed
organism. They are derived from the inner cell mass of early embryos (blastocysts)
[ 20- 23]. The cells in the inner cell mass of a given blastocyst are genetically
identical, and each blastocyst yields only a single ES cell line. Stem cells are rarer
[ 24] and more difficult to find in adults than in preimplantation embryos, and it has
proved harder to grow some kinds of adult stem cells into cell lines after isolation
[ 25; 26].

Production of different cells and tissues from ES cells or other stem cells is a subject
of current research [ 11; 27- 31]. Production of whole organs other than bone marrow
(to be used in bone marrow transplantation) from such cells has not yet been
achieved, and its eventual success is uncertain.

Current interest in stem cells arises from their potential for the therapeutic
transplantation of particular healthy cells, tissues, and organs into people suffering
from a variety of diseases and debilitating disorders. Research with adult stem cells
indicates that they may be useful for such purposes, including for tissues other than
those from which the cells were derived [ 12; 14; 17; 18; 25- 27; 32- 43]. On the basis
of current knowledge, it appears unlikely that adults will prove to be a sufficient
source of stem cells for all kinds of tissues [ 11; 44- 47]. ES cell lines are of potential
interest for transplantation because one cell line can multiply indefinitely and can
generate not just one type of specialized cell, but many different types of specialized
cells (brain, muscle, and so on) that might be needed for transplants
[ 20; 28; 45; 48; 49]. However, much more research will be needed before the
magnitude of the therapeutic potential of either adult stem cells or ES cells will be
well understood.

One of the most important questions concerning the therapeutic potential of stem cells
is whether the cells, tissues, and perhaps organs derived from them can be
transplanted with minimal risk of transplant rejection. Ideally, adult stem cells
advantageous for transplantation might be derived from patients themselves. Such
cells, or tissues derived from them, would be genetically identical with the patient's
own and not be rejected by the immune system. However, as previously described, the
availability of sufficient adult stem cells and their potential to give rise to a full range
of cell and tissue types are uncertain. Moreover, in the case of a disorder that has a
genetic origin, a patient's own adult stem cells would carry the same defect and would
have to be grown and genetically modified before they could be used for therapeutic
transplantation.

The application of somatic cell nuclear transfer or nuclear transplantation offers an

alternative route to obtaining stem cells that could be used for transplantation
therapies with a minimal risk of transplant rejection. This procedure—sometimes
called therapeutic cloning, research cloning, or nonreproductive cloning, and referred
to here as nuclear transplantation to produce stem cells—would be used to
generate pluripotent ES cells that are genetically identical with the cells of a
transplant recipient [ 50]. Thus, like adult stem cells, such ES cells should ameliorate
the rejection seen with unmatched transplants.

Two types of adult stem cells—stem cells in the blood forming bone marrow and skin
stem cells—are the only two stem cell therapies currently in use. But, as noted in the
National Academies' report entitled Stem Cells and the Future of Regenerative
Medicine, many questions remain before the potential of other adult stem cells can be
accurately assessed [ 11]. Few studies on adult stem cells have sufficiently defined the
stem cell's potential by starting from a single, isolated cell, or defined the necessary
cellular environment for correct differentiation or the factors controlling the efficiency
with which the cells repopulate an organ. There is a need to show that the cells
derived from introduced adult stem cells are contributing directly to tissue function,
and to improve the ability to maintain adult stem cells in culture without the cells
differentiating. Finally, most of the studies that have garnered so much attention have
used mouse rather than human adult stem cells.

ES cells are not without their own potential problems as a source of cells for
transplantation. The growth of human ES cells in culture requires a “feeder” layer of
mouse cells that may contain viruses, and when allowed to differentiate the ES cells
can form a mixture of cell types at once. Human ES cells can form benign tumors
when introduced into mice [ 20], although this potential seems to disappear if the cells
are allowed to differentiate before introduction into a recipient [ 51]. Studies with
mouse ES cells have shown promise for treating diabetes [ 30], Parkinson's disease
[ 52], and spinal cord injury [ 53].

The ES cells made with nuclear transplantation would have the advantage over adult
stem cells of being able to provide virtually all cell types and of being able to be
maintained in culture for long periods of time. Current knowledge is, however,
uncertain, and research on both adult stem cells and stem cells made with nuclear
transplantation is required to understand their therapeutic potentials. (This point is
stated clearly in Finding and Recommendation 2 of Stem Cells and the Future of
Regenerative Medicine [ 11] which states, in part, that “studies of both embryonic and
adult human stem cells will be required to most efficiently advance the scientific and
therapeutic potential of regenerative medicine.”) It is likely that the ES cells will
initially be used to generate single cell types for transplantation, such as nerve cells or
muscle cells. In the future, because of their ability to give rise to many cell types, they
might be used to generate tissues and, theoretically, complex organs for
transplantation. But this will require the perfection of techniques for directing their
specialization into each of the component cell types and then the assembly of these
cells in the correct proportion and spatial organization for an organ. That might be
reasonably straightforward for a simple structure, such as a pancreatic islet that
produces insulin, but it is more challenging for tissues as complex as that from lung,
kidney, or liver [ 54; 55].

The experimental procedures required to produce stem cells through nuclear

transplantation would consist of the transfer of a somatic cell nucleus from a patient
into an enucleated egg, the in vitro culture of the embryo to the blastocyst stage, and
the derivation of a pluripotent ES cell line from the inner cell mass of this blastocyst.
Such stem cell lines would then be used to derive specialized cells (and, if possible,
tissues and organs) in laboratory culture for therapeutic transplantation. Such a
procedure, if successful, can avoid a major cause of transplant rejection. However,
there are several possible drawbacks to this proposal. Experiments with animal
models suggest that the presence of divergent mitochondrial proteins in cells may
create “minor” transplantation antigens [ 56; 57] that can cause rejection [ 58- 63];
this would not be a problem if the egg were donated by the mother of the transplant
recipient or the recipient herself. For some autoimmune diseases, transplantation of
cells cloned from the patient's own cells may be inappropriate, in that these cells can
be targets for the ongoing destructive process. And, as with the use of adult stem cells,
in the case of a disorder that has a genetic origin, ES cells derived by nuclear
transplantation from the patient's own cells would carry the same defect and would
have to be grown and genetically modified before they could be used for therapeutic
transplantation. Using another source of stem cells is more likely to be feasible
(although immunosuppression would be required) than the challenging task of
correcting the one or more genes that are involved in the disease in adult stem cells or
in a nuclear transplantation-derived stem cell line initiated with a nucleus from the
patient.

In addition to nuclear transplantation, there are two other methods by which

researchers might be able to derive ES cells with reduced likeli hood for rejection. A
bank of ES cell lines covering many possible genetic makeups is one possibility,
although the National Academies report entitled Stem Cells and the Future of
Regenerative Medicine rated this as “difficult to conceive” [ 11]. Alternatively,
embryonic stem cells might be engineered to eliminate or introduce certain cell-
surface proteins, thus making the cells invisible to the recipient's immune system. As
with the proposed use of many types of adult stem cells in transplantation, neither of
these approaches carries anything close to a promise of success at the moment.

The preparation of embryonic stem cells by nuclear transplantation differs from

reproductive cloning in that nothing is implanted in a uterus. The issue of whether ES
cells alone can give rise to a complete embryo can easily be misinterpreted. The titles
of some reports suggest that mouse embryos can be derived from ES cells alone
[ 64- 72]. In all cases, however, the ES cells need to be surrounded by cells derived
from a host embryo, in particular trophoblast and primitive endoderm. In addition to
forming part of the placenta, trophoblast cells of the blastocyst provide essential
patterning cues or signals to the embryo that are required to determine the orientation
of its future head and rump (anterior-posterior) axis. This positional information is not
genetically determined but is acquired by the trophoblast cells from events initiated
soon after fertilization or egg activation. Moreover, it is critical that the positional
cues be imparted to the inner cells of the blastocyst during a specific time window of
development [ 73- 76]. Isolated inner cell masses of mouse blastocysts do not implant
by themselves, but will do so if combined with trophoblast vesicles from another
embryo [ 77]. By contrast, isolated clumps of mouse ES cells introduced into
trophoblast vesicles never give rise to anything remotely resembling a
postimplantation embryo, as opposed to a disorganized mass of trophoblast. In other
words, the only way to get mouse ES cells to participate in normal development is to
provide them with host embryonic cells, even if these cells do not remain viable
throughout gestation (Richard Gardner, personal communication). It has been
reported that human [ 20] and primate [ 78- 79] ES cells can give rise to trophoblast
cells in culture. However, these trophoblast cells would presumably lack the
positional cues normally acquired during the development of a blastocyst from an
egg. In the light of the experimental results with mouse ES cells described above, it is
very unlikely that clumps of human ES cells placed in a uterus would implant and
develop into a fetus. It has been reported that clumps of human ES cells in culture,
like clumps of mouse ES cells, give rise to disorganized aggregates known as
embryoid bodies [ 80].

Besides their uses for therapeutic transplantation, ES cells obtained by nuclear

transplantation could be used in laboratories for several types of studies that are
important for clinical medicine and for fundamental research in human developmental
biology. Such studies could not be carried out with mouse or monkey ES cells and are
not likely to be feasible with ES cells prepared from normally fertilized blastocysts.
For example, ES cells derived from humans with genetic diseases could be prepared
through nuclear transplantation and would permit analysis of the role of the mutated
genes in both cell and tissue development and in adult cells difficult to study
otherwise, such as nerve cells of the brain. This work has the disadvantage that it
would require the use of donor eggs. But for the study of many cell types there may
be no alternative to the use of ES cells; for these cell types the derivation of primary
cell lines from human tissues is not yet possible.

If the differentiation of ES cells into specialized cell types can be understood and
controlled, the use of nuclear transplantation to obtain genetically defined human ES
cell lines would allow the generation of genetically diverse cell lines that are not
readily obtainable from embryos that have been frozen or that are in excess of clinical
need in IVF clinics. The latter do not reflect the diversity of the general population
and are skewed toward genomes from couples in which the female is older than the
period of maximal fertility or one partner is infertile. In addition, it might be
important to produce stem cells by nuclear transplantation from individuals who have
diseases associated with both simple [81] and complex (multiple-gene) heritable
genetic predilections. For example, some people have mutations that predispose them
to “Lou Gehrig's disease” (amyotrophic lateral sclerosis, or ALS); however, only
some of these individuals become ill, presumably because of the influence of
additional genes. Many common genetic predilections to diseases have similarly
complex etiologies; it is likely that more such diseases will become apparent as the
information generated by the Human Genome Project is applied. It would be possible,
by using ES cells prepared with nuclear transplantation from patients and healthy
people, to compare the development of such cells and to study the fundamental
processes that modulate predilections to diseases.

Neither the work with ES cells, nor the work leading to the formation of cells and
tissues for transplantation, involves the placement of blastocysts in a uterus. Thus,
there is no embryonic development beyond the 64 to 200 cell stage, and no fetal
development.
 Go to:

Watch these videos of enucleation and nuclear transfer.

Cloning
Dolly the sheep, the first mammal cloned from a somatic (body) cell, came into the
world innocent as a lamb. However, soon after the announcement of her birth in
February 1997 (Wilmut et al., 1997) she caused panic and controversy. An important,
and for many people troubling question arose: if the cloning of sheep is possible, will
scientists soon start cloning humans as well; and if they did, would this be wrong or
unwise?

For most people, Dolly was really a wolf in sheep’s clothing. She represented a first
undesirable and dangerous step to applying reproductive cloning in humans,
something that many agreed should never be done. Only a small minority thought it
was permissible, or even morally obligatory to conduct further research into human
reproductive cloning. Some had no strong objections to it, but did not see any reason
to promote it either.

Dolly is now stuffed and set up for display in the National Museum of Scotland.
Many countries or jurisdictions have legally banned human cloning or are in the
process of doing so. In some countries, including France and Singapore, reproductive
cloning of humans is a criminal offence. In 2005, UNESCO adopted a ‘Declaration on
Human Cloning’, which calls for a universal ban on human cloning (for an
examination of the human cloning debate at UNESCO since 2008, see Langlois,
2017). The debate on human reproductive cloning seems to have drawn to a close.
However, since reproductive cloning of mammals has become routine in several
countries, there is reason to believe that at some point in the future, humans will be
cloned too. Moreover, even if reproductive cloning will not be possible in the near
future, cloning for research and therapeutic purposes is likely to be.

This entry describes the most important areas of disagreement regarding the ethics of
cloning. I will focus on human cloning (as opposed to animal cloning), since human
cloning has been the focus of the cloning debate.
  1. What is Cloning?
 2. Cloning for Research and Therapy
o 2.1 Creating and Killing Embryos for Stem Cells
o 2.2 The Need for Oocytes
o 2.3 Social Justice Considerations
o 2.4 A Slippery Slope to Reproductive Cloning
 3. Human Reproductive Cloning

o 3.1 Safety and Efficiency

o 3.2 Harm to the Individual Conceived through Cloning
o 3.3 Harm to Others
o 3.4 Human Dignity

 4. Religious perspectives
 Bibliography
 Academic Tools
 Other Internet Resources
 Related Entries

1. What is Cloning?
Strictly speaking, cloning is the creation of a genetic copy of a sequence of DNA or of
the entire genome of an organism. In the latter sense, cloning occurs naturally in the
birth of identical twins and other multiples. But cloning can also be done artificially in
the laboratory via embryo twinning or splitting: an early embryo is split in vitro so
that both parts, when transferred to a uterus, can develop into individual organisms
genetically identical to each other. In the cloning debate, however, the term ‘cloning’
typically refers to a technique called somatic cell nuclear transfer (SCNT). SCNT
involves transferring the nucleus of a somatic cell into an oocyte from which the
nucleus and thus most of the DNA has been removed. (The mitochondrial DNA in the
cytoplasm is still present). The manipulated oocyte is then treated with an electric
current in order to stimulate cell division, resulting in the formation of an embryo.
The embryo is (virtually) genetically identical to, and thus a clone of the somatic cell
donor.

Dolly was the first mammal to be brought into the world using SCNT. Ian Wilmut and
his team at the Roslin Institute in Scotland replaced the nucleus from an oocyte taken
from a Blackface ewe with the nucleus of a cell from the mammary gland of a six-
year old Finn Dorset sheep (these sheep have a white face). They transferred the
resulting embryo into the uterus of a surrogate ewe and approximately five months
later Dolly was born. Dolly had a white face: she was genetically identical to the Finn
Dorset ewe from which the somatic cell had been obtained.

Dolly, however, was not 100% genetically identical to the donor animal. Genetic
material comes from two sources: the nucleus and the mitochondria of a cell.
Mitochondria are organelles that serve as power sources to the cell. They contain
short segments of DNA. In Dolly’s case, her nuclear DNA was the same as the donor
animal; other of her genetic materials came from the mitochondria in the cytoplasm of
the enucleated oocyte. For the clone and the donor animal to be exact genetic copies,
the oocyte too would have to come from the donor animal (or from the same maternal
line – mitochondria are passed on by oocytes).

Dolly’s birth was a real breakthrough, for it proved that something that had been
considered biologically impossible could indeed be done. Before Dolly, scientists
thought that cell differentiation was irreversible: they believed that, once a cell has
differentiated into a specialized body cell, such as a skin or liver cell, the process
cannot be reversed. What Dolly demonstrated was that it is possible to take a
differentiated cell, turn back its biological clock, and make the cell behave as though
it was a recently fertilized egg.

Nuclear transfer can also be done using a donor cell from an embryo instead of from
an organism after birth. Cloning mammals using embryonic cells has been successful
since the mid-1980s (for a history of cloning, see Wilmut et al. 2001). Another
technique to produce genetically identical offspring or clones is embryo twinning or
embryo splitting, in which an early embryo is split in vitro so that both parts, when
implanted in the uterus, can develop into individual organisms genetically identical to
each other. This process occurs naturally with identical twins.

However, what many people find disturbing is the idea of creating a genetic duplicate
of an existing person, or a person who has existed. That is why the potential
application of SCNT in humans set off a storm of controversy. Another way to
produce a genetic duplicate from an existing person is by cryopreserving one of two
genetically identical embryos created in vitro for several years or decades before
using it to generate a pregnancy. Lastly, reproductive cloning of humans could, in
theory, also be achieved by combining the induced pluripotent stem cell technique
with tetraploid complementation. Several research teams have succeeded in cloning
mice this way (see, for example, Boland et al. 2009). The technique involves injecting
mouse iPS cells in tetraploid embryos, i.e. embryos with twice the normal number of
chromosomes that cannot result in live offspring. The resulting mouse pups are
derived solely from the iPS cells, which means that the tetraploid embryos only acted
as a substitute trophectoderm, which forms the placenta and other nourishing
membranes but which does not contribute to the ‘embryo proper’.

Dolly is a case of reproductive cloning, the aim of which is to create offspring.

Reproductive cloning is to be distinguished from cloning for therapy and research,
sometimes also referred to as ‘therapeutic cloning’. Both reproductive cloning and
cloning for research and therapy involve SCNT, but their aims, as well as most of the
ethical concerns they raise, differ. I will first discuss cloning for research and therapy
and will then proceed to outline the ethical debate surrounding reproductive cloning.
2. Cloning for Research and Therapy
Cloning for research and therapy involves the creation of an embryo via SCNT, but
instead of transferring the cloned embryo to the uterus in order to generate a
pregnancy, it is used to obtain pluripotent stem cells. It is thus not the intention to use
the embryo for reproductive purposes. Embryonic stem cells offer powerful tools for
developing therapies for currently incurable diseases and conditions, for important
biomedical research, and for drug discovery and toxicity testing (Cervera & Stojkovic
2007). For example, one therapeutic approach is to induce embryonic stem cells to
differentiate into cardiomyocytes (heart muscle cells) to repair or replace damaged
heart tissue, into insulin-producing cells to treat diabetes, or into neurons and their
supporting tissues to repair spinal cord injuries.

A potential problem with embryonic stem cells is that they will normally not be
genetically identical to the patient. Embryonic stem cells are typically derived from
embryos donated for research after in vitro fertilization (IVF) treatment. Because
these stem cells would have a genetic identity different from that of the recipient – the
patient – they may, when used in therapy, be rejected by her immune system.
Immunorejection can occur when the recipient’s body does not recognize the
transplanted cells, tissues or organs as its own and as a defense mechanism attempts
to destroy the graft. Another type of immunorejection involves a condition called
graft-versus-host disease, in which immune cells contaminating the graft recognize
the new host – the patient – as foreign and attack the host’s tissues and organs. Both
types of immunorejection can result in loss of the graft or death of the patient. It is
one of the most serious problems faced in transplant surgery.

Cloning for research and therapy could potentially offer a solution to this problem. An
embryo produced via SNCT using the patient’s somatic cell as a donor cell would be
virtually genetically identical to the patient. Stem cells obtained from that embryo
would thus also be genetically identical to the patient, as would be their derivatives,
and would be less likely to be rejected after transplantation. Though therapies using
embryonic stem cells from SCNT embryos are not yet on the horizon for humans,
scientists have provided proof of concept for these therapies in the mouse.

Embryonic stem cells from cloned embryos would also have significant advantages
for biomedical research, and for drug discovery and toxicity testing. Embryonic stem
cells genetically identical to the patient could provide valuable in vitro models to
study disease, especially where animal models are not available, where the research
cannot be done in patients themselves because it would be too invasive, or where
there are too few patients to work with (as in the case of rare genetic diseases).
Researchers could, for example, create large numbers of embryonic stem cells
genetically identical to the patient and then experiment on these in order to understand
the particular features of the disease in that person. The embryonic stem cells and
their derivatives could also be used to test potential treatments. They could, for
example, be used to test candidate drug therapies to predict their likely toxicity. This
would avoid dangerous exposure of patients to sometimes highly experimental drugs.

Cloning for research and therapy is, however, still in its infancy stages. In 2011, a
team of scientists from the New York Stem Cell Foundation Laboratory was the first
to have succeeded in creating two embryonic stem cell lines from human embryos
produced through SCNT (Noggle et al. 2011). Three years earlier, a small San Diego
biotechnological company created human embryos (at the blastocyst stage) via SCNT
but did not succeed in deriving embryonic stem cells from these cells (French et al.
2008). Cloning for research and therapy is thus not likely to bear fruit in the short
term though progress is made (Tachibana et al. 2013; Zhang et al. 2020). Apart from
unsolved technical difficulties, much more basic research in embryonic stem cell
research is needed. The term ‘therapeutic cloning’ has been criticized precisely for
this reason. It suggests that therapy using embryonic stem cells from cloned embryos
is already reality. In the phase before clinical trials, critics say, it is only reasonable to
refer to research on nuclear transfer as ‘research cloning’ or ‘cloning for biomedical
research’ (PCBE, 2002).

Cloning for research and therapy holds great potential for future research and
therapeutic applications, but it also raises various ethical concerns.

2.1 Creating and Killing Embryos for Stem Cells

Much of the debate about the ethics of cloning for research and therapy turns on a
basic disagreement about how we should treat early human embryos. As it is currently
done, the isolation of embryonic stem cells involves the destruction of embryos at the
blastocyst stage (day five after fertilization, when the embryo consists of 125–225
cells). But cloning for research and therapy not only involves the destruction of
embryos, it also involves the creation of embryos solely for the purpose of stem cell
derivation. Views on whether and when it is permissible to create embryos solely to
obtain stem cells differ profoundly.

Some believe that an embryo, from the moment of conception, has the same moral
status, that is, the same set of basic moral rights, claims or interests as an ordinary
adult human being. This view is sometimes expressed by saying that the early embryo
is a person. On this view, creating and killing embryos for stem cells is a serious
moral wrong. It is impermissible, even if it could save many lives (Deckers 2007).
Others believe that the early embryo is merely a cluster of cells or human tissue
lacking any moral status. A common view among those who hold this position is that,
given its promising potential, embryonic stem cell and cloning research is a moral
imperative (Devolder & Savulescu 2006). Many defend a view somewhere in between
these opposing positions. They believe, for example, that the early embryo should be
treated with respect because it has an intermediate moral status: a moral status lower
than that of a person but higher than that of an ordinary body cell. A popular view
amongst those who hold this position is that using embryos for research might
sometimes be justified. Respect can be demonstrated, it is typically argued, by using
embryos only for very important research that cannot be done using less controversial
means, and by acknowledging the use of embryos for research with a sense of regret
or loss (Robertson 1995; Steinbock 2001). One common view among those who hold
the intermediate moral status view is that the use of discarded IVF embryos to obtain
stem cells is compatible with the respect we owe to the embryo, whereas the creation
and use of cloned embryos is not. An argument underlying this view is that, unlike
IVF embryos, cloned embryos are created for instrumental use only; they are created
and treated as a mere means, which some regard as incompatible with respectful
treatment of the embryo (NBAC 1999). Others (both proponents and opponents of
embryo research) have denied that there is a significant moral difference between
using discarded IVF embryos and cloned embryos as a source of stem cells. They
have argued that if killing embryos for research is wrong, it is wrong regardless of the
embryo’s origin (Doerflinger 1999; Fitzpatrick 2003; Devolder 2005, 2015). Douglas
and Savulescu (2009) have argued that it is permissible to destroy ‘unwanted’
embryos in research, that is, embryos that no one wishes to use for reproductive
purposes. Since both discarded IVF embryos and cloned embryos created for the
purpose of stem cell derivation are unwanted embryos in that sense, it is, on their
view, permissible to use both types of embryos for research.

A less common view holds that obtaining stem cells from cloned embryos
poses fewer ethical problems than obtaining stem cells from discarded IVF embryos.
Hansen (2002) has advanced this view, arguing that embryos resulting from SCNT do
not have the same moral status we normally accord to other embryos: he calls the
combination of a somatic nucleus and an enucleated egg a “transnuclear egg”, which,
he says, is a mere “artifact” with no “natural purpose” or potential “to evolve into an
embryo and eventually a human being,” and therefore falls outside the category of
human beings. McHugh (2004) and Kiessling (2001) advance a similar argument. On
their view, obtaining stem cells from cloned embryos is less morally problematic
because embryos resulting from SCNT cannot (yet) develop further, and are thus
better thought of as tissue culture, whereas IVF represents instrumental support for
human reproduction. Since creating offspring is not the goal, they argue, it is
misleading to use the term ‘embryo’ or ‘zygote’ to refer to the product of SCNT. They
suggest to instead use the terms ‘clonote’ (Mc Hugh) and ‘ovasome’ (Kiessling).

2.2 The Need for Oocytes

Cloning for research and therapy requires a large number of high-quality donor
oocytes. Ethical issues arise regarding how these oocytes could be obtained. Oocyte
donation involves various risks and discomforts (for a review of the risks, see
Committee on Assessing the Medical Risks of Human Oocyte Donation for Stem Cell
Research, 2007). Among the most pressing ethical issues raised by participating in
such donation is what model of informed consent should be applied. Unlike women
who are considering IVF, non-medical oocyte donors are not clinical patients. They
do not stand to derive any reproductive or medical benefit themselves from the
donation (though Kalfoglou & Gittelsohn, 2000, argue that they may derive a
psychological benefit). Magnus and Cho (2005) have argued that donating women
should not be classified as research subjects since, unlike in other research, the risks
to the donor do not lie in the research itself but in the procurement of the materials
required for the research. They suggest that a new category named ‘research donors’
be created for those who expose themselves to substantial risk only for the benefit of
others (in this case unidentifiable people in the future) and where the risk is incurred
not in the actual research but in the procurement of the materials for the research.
Informed consent for altruistic organ donation by living donors to strangers has also
been suggested as a model, since, in both cases, the benefits will be for strangers and
not for the donor. Critics of this latter suggestion have pointed out, however, that
there is a disanalogy between these two types of donation. The general ethical rule
reflected in regulations concerning altruistic donation, namely that there must be a
high chance of a good outcome for the patient, is violated in the case of oocyte
donation for cloning research (George 2007).
Given the risks to the donor, the absence of direct medical benefit for the donor, and
the uncertain potential of cloning research, it is not surprising that the number of
altruistic oocyte donations for such research is very low. Financial incentives might
be needed to increase the supply of oocytes for cloning research. In some countries,
including the US, selling and buying oocytes is legal. Some object to these practices
because they consider oocytes as integral to the body and think they should be kept
out of the market: on their view, the value of the human body and its parts should not
be expressed in terms of money or other fungible goods. Some also worry that,
through commercialization of oocytes, women themselves may become objects of
instrumental use (Alpers &Lo 1995). Many agree, however, that a concern for
commodification does not justify a complete ban on payment of oocyte donors and
that justice requires that they be financially compensated for the inconvenience,
burden, and medical risk they endure, as is standard for other research subjects
(Steinbock 2004; Mertes &Pennings 2007). A related concern is the effect of financial
or other offers of compensation on the voluntariness of oocyte donation. Women,
especially economically disadvantaged women from developing countries, might be
unduly induced or even coerced into selling their oocytes (Dickinson 2002). Baylis
and McLeod (2007) have highlighted how difficult it is concomitantly to avoid both
undue inducement and exploitation: a price that is too low risks exploitation; a price
that avoids exploitation risks undue inducement.

Concerns about exploitation are not limited to concerns about payment, as became
clear in the ‘Hwang scandal’ (for a review, see Saunders & Savulescu 2008). In 2004,
Woo-Suk-Hwang, a leading Korean stem cell scientist, claimed to be the first to clone
human embryos using SCNT and to extract stem cells from these embryos. In addition
to finding that Hwang had fabricated many of his research results, Korea’s National
Bioethics Committee also found that Hwang had pressured junior members of his lab
to donate oocytes for his cloning experiments.

Some authors have argued that a regulated market in oocytes could minimize ethical
concerns raised by the commercialization of oocytes and could be consistent with
respect for women (Resnik 2001; Gruen 2007). Researchers are also investigating the
use of alternative sources of oocytes, including animal oocytes, fetal oocytes, oocytes
from adult ovaries obtained post mortem or during operation, and stem cell-derived
oocytes. Scientists have already succeeded in creating human oocytes from embryonic
stem cells (Ma et al. 2017; Saitou & Miyauchi 2016). Finally, another option is ‘egg-
sharing’ where couples who are undergoing IVF for reproductive purposes have the
option to donate one or two of their oocytes in return for a reduced fee for their
fertility treatment. The advantage of this system is that it avoids exposing women to
extra risks – these women were undergoing IVF in any case (Roberts & Throsby
2008).

2.3 Social Justice Considerations

Personalized cloning therapies are likely to be labor intensive and expensive. This has
raised social justice concerns. Perhaps cloning therapies will only be a realistic option
for the very rich? Some have replied to this concern by pointing out that Cloning
therapies may become cheaper, less labor intensive and more widely accessible after
time. Moreover, cloning may cure diseases and not only treat symptoms. Regardless
of the economic cost, it remains true of course that the cloning procedure is time
consuming, rendering it inappropriate for certain clinical applications where urgent
intervention is required (e.g., myocardial infarction, acute liver failure or traumatic or
infectious spinal cord damage). If cloning for therapy became available, its
application would thus likely be restricted to chronic conditions. Wilmut (1997), who
cloned Dolly, has suggested that cloning treatments could be targeted to maximize
benefit: an older person with heart disease could be treated with stem cells that are not
a genetic match, take drugs to suppress her immune system for the rest of her life, and
live with the side-effects; a younger person might benefit from stem cells from cloned
embryos that match exactly. Devolder and Savulescu (2006) have argued that
objections about economic cost are most forceful against ‘cloning for self-
transplantation’ than, for example, against cloning for developing cellular models of
human disease. The latter will enable research into human diseases and may result in
affordable therapies and cures for a variety of common diseases, such as cancer and
heart disease, which afflict people all over the world. Finally, some have pointed out
that it is not clear whether cloning research is necessarily more labor intensive than
experiments on cells and tissues now done in animals.

Some are skeptical about the claimed benefits of cloning for research and therapy.
They stress that for many diseases in which cloned embryonic stem cells might offer a
therapy, there are alternative treatments and/or preventive measures in development,
including gene therapy, pharmacogenomical solutions and treatments based on
nanotechnology. It is often claimed that other types of stem cells such as adult stem
cells and stem cells from the umbilical cord blood might enable us to achieve the
same aims as cloning. Especially induced pluripotent stem cells (iPSCs) have raised
the hope that cloning research is superfluous (Rao & Condic 2008). iPSCs are created
through genetic manipulation of a body cell. iPSCs are similar to embryonic stem
cells, and in particular to embryonic stem cells from cloned embryos. However, iPSC
research could provide tissue- and patient-specific cells without relying on the need
for human oocytes or the creation and destruction of embryos. iPSC research could
thus avoid the ethical issues raised by cloning. This promise notwithstanding,
scientists have warned that it would be premature to stop cloning research as iPSCs
are not identical to embryonic stem cells (Pera & Trounson 2013). Cloning research
may teach us things that iPSC research cannot teach us. Moreover, iPSC research has
been said to fail to completely avoid the issue of embryo destruction (Brown 2009,
Devolder 2015).

2.4 A Slippery Slope to Reproductive Cloning

Slippery slope arguments express the worry that permitting a certain practice may
place us on a slippery slope to a dangerous or otherwise unacceptable outcome.
Several commentators have argued that accepting or allowing cloning research is the
first step that would place us on a slippery slope to reproductive cloning. As Leon
Kass (1998, 702) has put it: “once the genies put the cloned embryos into the bottles,
who can strictly control where they go?”

Others are more skeptical about slippery slope arguments against cloning and think
that effective legislation can prevent us from sliding down the slope (Savulescu 1999;
Devolder & Savulescu 2006). If reproductive cloning is unacceptable, these critics
say, it is reasonable to prohibit this specific technology rather than to ban non-
reproductive applications of cloning. The UK and Belgium, for example, allow
cloning research but prohibit the transfer of cloned embryos to the uterus.

Apart from the question of how slippery the slope might be, another question raised
by such arguments concerns the feared development –reproductive cloning– and
whether it is really ethically objectionable. Profound disagreement exists about the
answer to this question.

3. Human Reproductive Cloning

The central argument in favor of reproductive cloning is expansion of opportunities
for reproduction. Reproductive cloning could offer a new means for prospective
parents to satisfy their reproductive goals or desires. Infertile individuals or couples
could have a child that is genetically related to them. In addition, individuals, same
sex couples, or couples who cannot together produce an embryo would no longer need
donor gametes to reproduce if cloning were available (some might still need donor
eggs for the cloning procedure, but these would be enucleated so that only the
mitochondrial DNA remains). It would then be possible to avoid that one’s child
shares half of her nuclear DNA with a gamete donor.

Using cloning to help infertile people to have a genetically related child, or a child
that is only genetically related to them, has been defended on the grounds of human
wellbeing, personal autonomy, and the satisfaction of the natural inclination to
produce offspring (Häyry 2003; Strong 2008). Offering individuals or couples the
possibility to reproduce using cloning technology has been said to be consistent with
the right to reproductive freedom, which, according to some, implies the right to
choose what kind of children we will have (Brock 1998, 145).

According to some, the main benefit of reproductive cloning is that it would enable
prospective parents to control what genome their children will be endowed with
(Fletcher 1988, Harris 1997, 2004; Pence 1998, 101–6; Tooley 1998). Cloning would
enable parents to have a child with a genome identical to that of a person with good
health and/or other desirable characteristics.

Another possible use of reproductive cloning is to create a child that is a tissue match
for a sick sibling. The stem cells from the umbilical cord blood or from the bone
marrow of the cloned child could be used to treat the diseased sibling. Such ‘saviour
siblings’, have already been created through sexual reproduction or, more efficiently,
through a combination of IVF, preimplantation genetic diagnosis and HLA testing.

Many people, however, have expressed concerns about human reproductive cloning.
For some, these concerns are sufficient to reject human cloning. For others, these
concerns should be weighed against reasons for reproductive cloning.

What follows is an outline of some of the main areas of concern and disagreement
about human reproductive cloning.

3.1 Safety and Efficiency

Despite the successful creation of viable offspring via SCNT in various mammalian
species, researchers still have limited understanding of how the technique works on
the subcellular and molecular level. Although the overall efficiency and safety of
reproductive cloning in mammals has significantly increased over the past fifteen
years, it is not yet a safe process (Whitworth & Prather 2010). For example, the rate
of abortions, stillbirths and developmental abnormalities remains high. Another
source of concern is the risk of premature ageing because of shortened telomeres.
Telomeres are repetitive DNA sequences at the tip of chromosomes that get shorter as
an animal gets older. When the telomeres of a cell get so short that they disappear, the
cell dies. The concern is that cloned animals may inherit the shortened telomeres from
their older progenitor, with possibly premature aging and a shortened lifespan as a
result.

For many, the fact that reproductive cloning is unsafe provides a sufficient reason not
to pursue it. It has been argued that it would simply be wrong to impose such
significant health risks on humans. The strongest version of this argument states that it
would be wrong now to produce a child using SCNT because it would constitute a
case of wrongful procreation. Some adopt a consent-based objection and condemn
cloning because the person conceived cannot consent to being exposed to significant
risks involved in the procedure (Kass 1998; PCBE 2002). Against this, it has been
argued that even if reproductive cloning is unsafe, it may still be permissible if there
are no safer means to bring that very same child into existence so long as the child is
expected to have a life worth living (Strong 2005).

With the actual rate of advancement in cloning, one cannot exclude a future in which
the safety and efficiency of SCNT will be comparable or superior to that of IVF or
even sexual reproduction. A remaining question is, then, whether those who condemn
cloning because of its experimental nature should continue to condemn it morally and
legally. Some authors have reasoned that if, in the future, cloning becomes safer than
sexual reproduction, we should even make it our reproductive method of choice
(Fletcher 1988; Harris 2004, Ch. 4).

3.2 Harm to the Individual Conceived through Cloning

3.2.1 A Threat to Autonomy

Some fear that cloning threatens the identity and individuality of the clone, thus
reducing her autonomy (Ramsey 1966; Kitcher 1997; Annas 1998; Kass 1998). This
may be bad in itself, or bad because it might reduce the clone’s wellbeing. It may also
be bad because it will severely restrict the array of life plans open to the clone, thus
violating her ‘right to an open future’ (a concept developed in Feinberg 1980). In its
report ‘Human Cloning and Human Dignity: An Ethical Inquiry’, the US President’s
Council on Bioethics (2002) wrote that being genetically unique is “an emblem of
independence and individuality” and allows us to go forward “with a relatively
indeterminate future in front of us” (Ch. 5, Section c). Such concerns have formed the
basis of strong opposition to cloning.

The concern that cloning threatens the clone’s identity and individuality has been
criticized for relying on the mistaken belief that who and what we become is entirely
determined by our genes. Such genetic determinism is clearly false. Though genes
influence our personal development, so does the complex and irreproducible context
in which our lives take place. We know this, among others, from studying
monozygotic twins. Notwithstanding the fact that such twins are genetically identical
to each other and, therefore, sometimes look very similar and often share many
character traits, habits and preferences, they are different individuals, with different
identities (Segal 2000). Thus, it is argued, having a genetic duplicate does not threaten
one’s individuality, or one’s distinct identity.

Brock (2002) has pointed out that one could nevertheless argue that even though
individuals created through cloning would be unique individuals with a distinct
identity, they might not experience it that way. What is threatened by cloning then is
not the individual’s identity or individuality, but her sense of identity and
individuality, and this may reduce her autonomy. So even if a clone has a unique
identity, she may experience more difficulties in establishing her identity than if she
had not been a clone.

But here too critics have relied on the comparison with monozygotic twins. Harris
(1997, 2004) and Tooley (1998), for example, have pointed out that each twin not
only has a distinct identity, but generally also views him or herself as having a distinct
identity, as do their relatives and friends. Moreover, so they argue, an individual
created through cloning would likely be of a different age than her progenitor. There
may even be several generations between them. A clone would thus in essence be a
‘delayed’ twin. Presumably this would make it even easier for the clone to view
herself as distinct from the progenitor than if she had been genetically identical to
someone her same age.

However, the reference to twins as a model to think about reproductive cloning has
been criticized, for example, because it fails to reflect important aspects of the parent-
child relationship that would incur if the child were a clone of one of the rearing
parents (Jonas 1974; Levick 2004). Because of the dominance of the progenitor, the
risk of reduced autonomy and confused identity may be greater in such a situation
than in the case of ordinary twins. Moreover, just because the clone would be a
delayed twin, she may have the feeling that her life has already been lived or that she
is predetermined to do the same things as her progenitor (Levy & Lotz 2005). This
problem may be exacerbated by others constantly comparing her life with that of the
progenitor, and having problematic expectations based on these comparisons. The
clone may feel under constant pressure to live up to these expectations (Kass 1998;
Levick 2004, 101; Sandel 2007, 57–62), or may have the feeling she leads ‘a life in
the shadow’ of the progenitor (Holm 1998; PCBE 2002, Ch.5). This may especially
be the case if the clone was created as a ‘replacement’ for a deceased child. (Some
private companies already offer to clone dead pets to create replacements pets.) The
fear is that the ‘ghost of the dead child’ will get more attention and devotion than the
replacement child. Parents may expect the clone to be like the lost child, or some
idealized image of it, which could hamper the development of her identity and
adversely affect her self-esteem (Levick 2004, 111–132). Finally, another reason why
the clone’s autonomy may be reduced is because she would be involuntarily informed
about her genetic predispositions. A clone who knows that her genetic parent
developed a severe single gene disease at the age of forty will realise it is very likely
that she will undergo the same fate. Unlike individuals who choose to have
themselves genetically tested, clones who know their genetic parent’s medical history
will be involuntarily informed.

These concerns have been challenged on several grounds. Some believe that it is
plausible that, through adequate information, we could largely correct mistaken
beliefs about the link between genetic and personal identity, and thus reduce the risk
of problematic expectations toward the clone (Harris 1997, 2004; Tooley 1998, 84–5;
Brock 1998, Pence 1998). Brock (1998) and Buchanan et al. (2000, 198) have argued
that even if people persist in these mistaken beliefs and their attitudes or actions lead
to cloned individuals believing they do not have an open future, this does not imply
that the clone’s right to ignorance about one’s personal future or to an open future
has actually been violated. Pence (1998, 138) has argued that having high
expectations, even if based on false beliefs, is not necessarily a bad thing. Parents
with high expectations often give their children the best chances to lead a happy and
successful life. Brock (2002, 316) has argued that parents now also constantly restrict
the array of available life plans open to their children, for example, by selecting their
school or by raising them according to certain values. Though this may somewhat
restrict the child’s autonomy, there will always be enough decisions to take for the
child to be autonomous, and to realize this. According to Brock, it is not clear why
this should be different in the case of cloning. He also points out that there may be
advantages to being a ‘delayed twin’ (154). For example, one may acquire knowledge
about the progenitor’s medical history and use this knowledge to live longer, or to
increase one’s autonomy. One could, for example, use the information to reduce the
risk of getting the disease or condition, or to at least postpone its onset, by behavioral
changes, an appropriate diet and/or preventive medication. This would not be
possible, however, if the disease is untreatable (for example, Huntington’s Disease).
Harris (2004, Ch.1) has stressed that information about one’s genetic predispositions
for certain diseases would also allow one to take better informed reproductive
decisions. Cloning would allow us to give our child a ‘tried and tested’ genome, not
one created by the genetic lottery of sexual reproduction and the random combination
of chromosomes.
3.2.2 The clone will be treated as a means

Cloning arouses people’s imagination about the clone, but also about those who will
choose to have a child through cloning. Often dubious motives are ascribed to them:
they would want a child that is ‘just like so-and-so’ causing people to view children as
objects or as commodities like a new car or a new house (Putnam 1997, 7–8). They
would want an attractive child (a clone of Scarlett Johansson) or a child with tennis
talent (a clone of Victoria Azarenka) purely to show off. Dictators would want armies
of clones to achieve their political goals. People would clone themselves out of
vanity. Parents would clone their existing child so that the clone can serve as an organ
bank for that child, or would clone their deceased child to have a replacement child.
The conclusion is then that cloning is wrong because the clone will be used as a mere
means to others’ ends. These critiques have also been expressed with regard to other
forms of assisted reproduction; but some worry that individuals created through
cloning may be more likely to be viewed as commodities because their total genetic
blueprint would be chosen – they would be “fully made and not begotten” (Ramsey
1966; Kass 1998; PCBE 2002, 107).
Strong (2008) has argued that these concerns are based on a fallacious inference. It is
one thing to desire genetically related children, and something else to believe that one
owns one’s children or that one considers one’s children as objects, he writes. Other
commentators, however, have pointed out that even if parents themselves do not
commodify their children, cloning might still have an impact on society as a whole,
thereby increasing the tendency of others to do so (Levy & Lotz 2005; Sandel 2007).
A related concern expressed by Levick (2004, 184–5) is that allowing cloning might
result in a society where ‘production on demand’ clones are sold for adoption to
people who are seeking to have children with special abilities – a clearer case of
treating children as objects.

But suppose some people create a clone for instrumental reasons, for example, as a
stem cell donor for a sick sibling. Does this imply that the clone will be treated merely
as a means? Critics of this argument have pointed out that parents have children for
all kinds of instrumental reasons, including the benefit for the husband-wife
relationship, continuity of the family name, and the economic and psychological
benefits children provide when their parents become old (Harris 2004, 41–2, Pence
1998). This is generally not considered problematic as long as the child is also valued
in its own right. What is most important in a parent-child relationship is the love and
care inherent in that relationship. They stress the fact that we judge people on their
attitudes toward children, rather than on their motives for having them. They also
deny that there is a strong link between one’s intention or motive to have a child, and
the way one will treat the child.
3.2.3 Societal Prejudice and Respect for Clones

Another concern is that clones may be the victims of unjustified discrimination and
will not be respected as persons (Deech 1999; Levick 2004, 185–187). Savulescu
(2005, Other Internet Resources) has referred to such negative attitudes towards
clones as ‘clonism’: a new form of discrimination against a group of humans who are
different in a non-morally significant way. But does a fear for ‘clonism’ constitute a
good reason for rejecting cloning? Savulescu and others have argued that, if it is, then
we must conclude that racist attitudes and discriminatory behavior towards people
with a certain ethnicity provides a good reason for people with that ethnicity not to
procreate. This, according to these critics, is a morally objectionable way to solve the
problem of racism. Instead of limiting people’s procreative liberty we should combat
existing prejudices and discrimination. Likewise, it is argued, instead of prohibiting
cloning out of concern for clonism, we should combat possible prejudices and
discrimination against clones (see also Pence 1998, 46; Harris 2004, 92–93).
Macintosh (2005, 119–21) has warned that by expressing certain concerns about
cloning one may actually reinforce certain prejudices and misguided stereotypes about
clones. For example, saying that a clone would not have a personal identity prejudges
the clone as inferior or fraudulent (the idea that originals are more valuable than their
copies) or even less than human (as individuality is seen as an essential characteristic
of human nature).
3.2.4 Complex Family Relationships

Some worry that cloning will threaten traditional family structures; a fear that has
come up in debates about gay people adopting children, IVF and other assisted
reproduction techniques. But in cloning the situation would be more complex as it
may blur generational boundaries (McGee 2000) and the clone would likely be
confused about her kinship ties (Kass 1998; O’Neil 2002, 67–68). For example, a
woman who has a child conceived through cloning would actually be the twin of her
child and the woman’s mother would, genetically, be its mother, not grandmother.
Some have argued against these concerns, replying that a cloned child would not
necessarily be more confused about her family ties than other children. Many have
four nurturing parents because of a divorce, never knew their genetic parents, have
nurturing parents that are not their genetic parents, or think that their nurturing father
is also their genetic father when in fact he is not. While these complex family
relationships can be troubling for some children, they are not insurmountable, critics
say. Harris (2004, 77–78) argues that there are many aspects about the situation one is
born and raised in that may be troublesome. As with all children, the most important
thing is the relation with people who nurture and educate them, and children usually
know very well who these people are. There is no reason to believe that with cloning,
this will be any different. Onora O’Neil (2002, 67–8) argues that such responses are
misplaced. While she acknowledges that there are already children now with confused
family relationships, she argues that it is very different when prospective parents seek
such potentially confused relationships for their children from the start.

3.3 Harm to Others

Other concerns related to cloning focus on the potential harmful effects of cloning for
others. Sometimes these concerns are related to those about the wellbeing of the
clone. For example, McGee’s concern about confused family relationships not only
bears on the clone but also on society as a whole. However, since I have already
mentioned this concern, I will, in the remainder of this entry, focus on other
arguments
3.3.1 Adoption and the Importance of Genetic Links

It is often claimed that the strongest reason for why reproductive cloning should be
permissible, if safe, is that it will allow infertile people to have a genetically related
child. This position relies on the view that having genetically related children is
morally significant and valuable. This is a controversial view. For example, Levy and
Lotz (2005) and Rulli (2016) have denied the importance of a genetic link between
parents and their children. Moreover, they have argued that claiming that this link is
important will give rise to bad consequences, such as reduced adoption rates (and, in
Rulli’s case, a failure to fulfil one’s duty to adopt) and diminished resources for
improving the life prospects of the disadvantaged, including those waiting to be
adopted. Levick (2004, 185) and Ahlberg and Brighouse (2011) have also advanced
this view. Since, according to these authors, these undesirable consequences would be
magnified if we allowed human cloning, we have good reason to prohibit it. In
response, Strong (2008) has argued that this effect is uncertain, and that there are
other, probably more effective, ways to help such children or to prevent them from
ending up in such a situation. Moreover, if cloning is banned, infertile couples may
make use of donor embryos or gametes rather than adoption. Rob Sparrow (2006) has
pointed out another potential problem for those who defend reproductive cloning for
the reason that it will overcome infertility by providing a genetically related child.
According to Sparrow, cloning just doesn’t provide the right sort of genetic relation to
make those who use the technology the parents of the child.So, in order to justify
reproductive cloning one then has to emphasise the importance of the intention with
which the parents bring the cloned child into the world, rather than the genetic
relationship with the child. And this emphasis works to undermine the justification for
reproductive cloning in the first place.
3.3.2 Genetic Diversity

Another concern is that because cloning is an asexual way of reproducing it would

decrease genetic variation among offspring and, in the long run, might even constitute
a threat to the human race. The gene pool may narrow sufficiently to threaten
humanity’s resistance to disease (AMA 1999, 6). In response, it has been argued that
if cloning becomes possible, the number of people who will choose it as their mode of
reproduction will very likely be too low to constitute a threat to genetic diversity. It
would be unlikely to be higher than the rate of natural twinning, which, occurring at a
rate of 3.5/1000 children, does not seriously impact on genetic diversity. Further, even
if millions of people would create children through cloning, the same genomes will
not be cloned over and over: each person would have a genetic copy of his or her
genome, which means the result will still be a high diversity of genomes. Others argue
that, even if genetic diversity were not diminished by cloning, a society that supports
reproductive cloning might be taken to express the view that variety is not important.
Conveying such a message, these authors say, could have harmful consequences for
society.
3.3.3 Eugenics

Some see the increase in control of what kind of genome we want to pass on to our
children as a positive development. A major concern, however, is that this shift ‘from
chance to choice’ will lead to problematic eugenic practices.

One version of this concern states that cloning would, from the outset, constitute a
problematic form of eugenics. However, critics have argued that this is implausible:
the best explanations of what was wrong with immoral cases of eugenics, such as the
Nazi eugenic programs, are that they involved coercion and were motivated by
objectionable moral beliefs or false non-moral beliefs. This would not necessarily be
the case were cloning to be implemented now (Agar 2004; Buchanan 2007). Unlike
the coercive and state-directed eugenics of the past, new ‘liberal eugenics’ defends
values such as autonomy, reproductive freedom, beneficence, empathy and the
avoidance of harm (Agar, 2004). Enthusiasts of so-called ‘liberal eugenics’ are
interested in helping individuals to prevent or diminish the suffering and increase the
well-being of their children by endowing them with certain genes.

Another version of the eugenics concern points out the risk of a slippery slope: the
claim is that cloning will lead to objectionable forms of eugenics—for example,
coercive eugenics—in the future. After all, historical cases of immoral eugenics often
developed from earlier well intentioned and less problematic practices (for a history
of eugenics as well as an analysis of philosophical and political issues raised by
eugenics, see Kevles 1985 and Paul 1995). According to Sandel (2007, Ch.5), for
example, ‘liberal eugenics’ might imply more state compulsion than first appears: just
as governments can force children to go to school, they could require people to use
genetics to have ‘better’ children.
A related concern expressed by Sandel (2007, 52–7) is that cloning, and enhancement
technologies in general, may result in a society in which parents will not accept their
child for what it is, reinforcing an already existing trend of heavily managed, high-
pressure child-rearing or ‘hyper-parenting’. Asch and Wasserman (2005, 202) have
expressed a similar concern; arguing that having more control over what features a
child has can pose an “affront to an ideal of unconditioned devotion”. Another
concern, most often expressed by disability rights advocates, is that if cloning is used
to have ‘better’ children, it may create a more intolerant climate towards those with a
disability or a serious disease, and that such practices can express negative judgments
about people with disabilities. This argument has also been advanced in the debate
about selective abortion, prenatal testing, and preimplantation genetic diagnosis.
Disagreement exists about whether these effects are likely. For example, Buchanan et
al. (2002, 278) have argued that one can devalue disability while valuing existing
disabled people and that trying to help parents who want to avoid having a disabled
child does not imply that society should make no efforts to increase accessibility for
existing people with disabilities.

3.4 Human Dignity

UNESCO’s Universal Declaration on the Human Genome and Human Rights (1997)
was the first international instrument to condemn human reproductive cloning as a
practice against human dignity. Article 11 of this Declaration states: “Practices which
are contrary to human dignity, such as reproductive cloning of human beings, shall
not be permitted…” This position is shared by the World Health Organization, the
European Parliament and several other international instruments. Critics have pointed
out that the reference to human dignity is problematic as it is rarely specified how
human dignity is to be understood, whose dignity is at stake, and how dignity is
relevant to the ethics of cloning (Harris 2004, Ch.2, Birnbacher 2005, McDougall
2008,). Some commentators state that it is the copying of a genome which violates
human dignity (Kass 1998); others have pointed out that this interpretation could be
experienced as an offence to genetically identical twins, and that we typically do not
regard twins as a threat to human dignity (although some societies in the past did), nor
do we prevent twins from coming into existence. On the contrary, IVF, which
involves an increased ‘risk’ of having twins, is a widely accepted fertility treatment.

Human dignity is most often related to Kant’s second formulation of the Categorical
Imperative, namely the idea that we should never use a person merely as a means to
an end. I have, however, already discussed this concern in section 4.2.2.

4. Religious perspectives
No unified religious perspective on human cloning exists; indeed, there are a diversity
of opinions within each individual religious tradition. For an overview of the
evaluation of cloning by the main religious groups see, for example, Cole-Turner
(1997) and Walters (2004). For a specifically Jewish perspective on cloning, see, for
example, Lipschutz (1999), for an Islamic perspective, Sadeghi (2007) and for a
Catholic perspective, Doerflinger (1999).
Cloning Definition
Natural vs. Artificial Cloning
Cloning can be natural or artificial. Examples of cloning that occur naturally
are as follows:

 vegetative reproduction in plants, e.g. water hyacinth producing

multiple copies of genetically identical plants through apomixis
 binary fission in bacteria
 parthenogenesis in certain animals

Clones can also be produced through artificial means. Biotechnological

methods are employed to produce such clones.

Making multiple copies by manipulation procedures or biotechnology is

artificial cloning. It can be by:

 molecular cloning, where copies of specific gene fragments are

produced
 cellular cloning, where single-celled organisms with the exact genetic
content of the original cell are produced in cell cultures
 organism cloning, or reproductive cloning, where a multicellular clone is
created generally through somatic cell nuclear transfer

Molecular Cloning
This is the process by which copies of biomolecules, such as DNAs, are
produced. It is used to amplify a particular DNA fragment
containing target genes. Apart from the genes (coding sequences), it is also
used in making multiple copies of promoters, non-coding sequences, and
randomly fragmented DNA. The general steps in molecular cloning are as
follows:

 Fragmentation
 Ligation
 Transfection
 Screening or selection

In fragmentation, the DNA strand is fragmented to isolate the desired DNA

segment. This is then followed by ligation wherein the DNA fragments are
glued together to achieve the desired sequence. Transfection is when the
newly formed pieces of DNA are inserted into the cell. The transfected cells
are then cultured. Screening or selection proceeds by identifying the cells with
the new DNA. This could be done by using selection markers or by PCR,
restriction fragment analysis, and/or DNA sequencing.
Cloning A Single Cell
Cloning a cell means deriving a population of cells from a single cell. In single-
celled organisms such as bacterial cells and yeast cells, the process entails
getting a sample and then inoculating it to the culture medium.

How about cloning a cell from a multicellular organism? This is rather a more
complex procedure involving the use of cloning cylinders (rings). In essence,
the cloning cylinder (a sterile polystyrene ring) is dipped in grease and then
placed over an individual colony where cloned cells inside the ring can
eventually be produced and subsequently collected for transferring into a new
vessel.

Somatic-cell nuclear transfer is a form of cloning where stem cells are

cloned for research or for therapeutic purposes, but not for reproduction. The
stem cells are cloned and harvested for studying human diseases and thereby
used for finding a cure or understanding the pathobiology of the disease. So
far, this technique has been used for agriculture and for cloning animals, such
as sheep, cattle, goats, and pigs. It is therefore seen as a solution to save
endangered species from extinction.

Reproductive Cloning
Organism cloning (also called reproductive cloning) refers to the procedure of
creating a new multicellular organism that is genetically identical to another.
As already mentioned, cloning is a form of asexual reproduction. No sex cells
(gametes) are involved in the process. Because there is no need for a mate,
the parent organism reproduces relatively faster than organisms that
reproduce sexually. Nevertheless, the disadvantage of asexual means,
including cloning, is the decreased genetic diversity in the species. The low
genetic variation could essentially make the offspring be similarly predisposed
to environmental stressors whereby their parent is susceptible. They are
therefore at risk of being wiped out by a particular environmental condition
especially if their parent is susceptible to it.

Reproductive cloning in bacteria

A clone is genetically identical to its parent. This can be typically seen among
bacteria that reproduce by binary fission. See the figure below for the general
steps of binary fission.

Reproductive cloning in plants and animals

Apomixis (apomictic parthenogenesis) produces full clones of the mother as it

does not entail meiosis. This is in contrast to automictic parthenogenesis
wherein meiosis is involved and therefore the progeny is only a “half” clone of
the mother. Apomixis is more common in plants.
 Vegetative propagation is another form of cloning in plants. It is when a new
plant emerges from vegetative parts. Examples are as follows:

In animals, clones are reproduced by asexual means, such as

parthenogenesis and budding. Examples of animals reproducing by
parthenogenesis are aphids, rotifers, nematodes, certain lizards, snakes,
birds, sharks, reptiles, and amphibians. Some of them reproduce by
parthenogenesis either facultatively, which means they can also reproduce
sexually) or obligately, which means they have no other means to reproduce
but by parthenogenesis.

Watch this vid as an example of animal reproduction by parthenogenesis:

Budding, in turn, is the formation of an outgrowth from an organism. This

outgrowth is capable of developing into a new individual and is genetically the
same as the parent. It may stay attached or eventually split off from the
parent. Examples of animals capable of reproducing by budding are hydra,
corals, echinoderm larvae, and some acoel flatworms. Here is an example of
the budding process.

Cloning

Summary
Cloning describes the processes used to create an exact genetic replica of another cell,
tissue or organism. The copied material, which has the same genetic makeup as the
original, is referred to as a clone. The most famous clone was a Scottish sheep named
Dolly.

There are three different types of cloning:

 Gene cloning, which creates copies of genes or segments of DNA

 Reproductive cloning, which creates copies of whole animals
 Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these
cells to grow healthy tissue to replace injured or diseased tissues in the human body.
Molecular Biology and Genetic Engineering
A. Wesley Burks MD, in Middleton's Allergy: Principles and Practice, 2020

Gene Cloning

One of the most important developments in the field of recombinant DNA technology
has been the technique of gene cloning. The first step in the cloning of a specific gene
is the construction of a comprehensive collection of cloned DNA fragments, the DNA
library or gene library, which includes at least a fragment that contains a gene of
interest (see later). The cloning of genetic material begins with the insertion of a DNA
fragment that contains a gene of interest into the purified DNA genome of a self-
replicating element, generally a virus or a plasmid, and the propagation of this
chimeric DNA molecule in a host organism. The process of gene cloning leads to the
amplification of specific DNA fragments more than 1012-fold. This allows the
isolation and chemical characterization of specific DNA sequences. A virus or
plasmid used in this way is known as acloning vector. A cloning vector is a DNA
molecule that has the following characteristics: (1) it is capable of replicating
independently of the host chromosome; (2) an organism containing the vector can be
grown preferentially; and (3) additional DNA can be inserted into the vector. There
are two classes of vectors: the plasmid vectors and the phage vectors.

Plasmid Vectors.

Plasmids are small, circular molecules of double-stranded DNA derived from larger
plasmids that occur naturally in bacteria.68 Most plasmid-cloning vectors are
designed to replicate inE. coli.69 All of the enzymes required for replication of
the plasmid DNA are produced by a host bacterium. The classic example of plasmid
vector is pBR322, which was one of the first such vectors to be recognized. The three
important features of plasmid vectors are as follows:

Origin of replication. This origin permits the efficient replication

of plasmid to a large number of copies of cells, by the plasmid's
replicon, a region of approximately 1000 bp encoding the site at
which DNA replication is initiated.

Presence of selectable marker. Most plasmid vectors encode a gene

that confers bacterial resistance to antibiotic. This allows
selection of clones carrying the plasmid in the medium containing
antibiotic.
 •

Cloning, or restriction enzyme, cleavage site. All cloning vectors

must have at least one cloning site (a specific DNA sequence that
is recognized and cut by a restriction endonuclease), where the
foreign DNA is inserted.

Three classes of restriction enzymes bind to DNA at the recognition sequence and
hydrolyze the phosphodiester bond on both strands of DNA. Such restriction sites
usually have twofold symmetry; that is, the restriction sites are palindromic. Class II
restriction endonucleases, which recognize a DNA sequence of four to
eight nucleotides, are preferred for DNA technology. The restriction enzyme EcoRI,
isolated fromE. coli, cleaves DNA at the sequence 5′-GAATTC.69 The EcoRI scans
the plasmid until it finds the GAATTC sequence, where it hydrolyzes the
phosphodiester bond between deoxyguanosine and deoxyadenosine on both strands of
the DNA, creating a 4-bp (AATT) single-stranded overhang. Because EcoRI is
palindromic, the overhanging single-stranded ends (sticky ends) are complementary to
each other and can hybridize or anneal to each other by base pairing. Now the DNA to
be cloned (cleaved from its source by EcoRI) is inserted into a plasmid vector whose
DNA sequence has been cut by restriction endonuclease. The DNA fragment anneals
to the vector through DNA ligase, which catalyzes the covalent joining of the vector
DNA to the new piece of DNA (chimeric DNA). The gene (DNA fragment to be
cloned) now becomes a passenger on the vector molecule, ready to be introduced into
bacteria (DNA transformation).

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Cloning
T. Takala, in Encyclopedia of Applied Ethics (Second Edition), 2012

Introduction

Human reproductive cloning became an issue in 1997 when the birth of Dolly the
sheep, the first cloned mammal, was announced. People throughout the world rushed
to condemn human cloning as an absolute moral wrong, and a number of laws and
treaties were quickly drafted in this spirit. Cloning is one of those things that people
love to hate, and if asked about it on the street, the vast majority of Europeans and
Americans would probably say that cloning should be banned. Although there have
been some voices excited about the possibilities that cloning humans might open, the
majority of discussions on public, political, and academic fora have echoed
the denunciation of the practice. However, when asked about the reasons, people find
it surprisingly difficult to point their finger to the exact features that make cloning an
absolute moral wrong. This article introduces the reader to the main arguments that
have been presented against human (reproductive) cloning and to the few that have
been put forward in favor of it.

Cloning is a general term that refers to a number of techniques used for different
purposes. For legal and ethical purposes, with regard to humans, a distinction is
usually made between therapeutic and reproductive cloning. In therapeutic cloning,
the aim is to clone cells that make particular organs or types of tissue – the most
promising uses are believed to be in stem cells, but cloning could also be used to
produce organs for transplantation. In reproductive cloning, the aim is to produce new
human beings. Although therapeutic cloning has also been perceived as ethically
problematic, it is far better tolerated than the idea of producing new human beings by
similar methods.

There are roughly two known ways of cloning mammals. The less controversial
method for human reproductive cloning is called embryo splitting. This happens
naturally when one embryo spontaneously divides into two or more embryos, thus
creating identical twins or, sometimes, triplets or even more. In its artificial form, an
existing embryo is mechanically divided into two or more embryos that are then
allowed to develop naturally. This method has been used with human embryos in
fertility clinics since 1993 and it is approved, for instance, by the American Medical
Association, but there are many countries in which it remains illegal. The technique
that has raised more moral outrage is the possibility of creating clones by nuclear
transfer. This is how Dolly was produced. In this method, by a process known as cell
fusion, the nucleus of a cell from another being (in Dolly’s case, a cell of an adult
sheep) is transferred into an unfertilized egg taken from a donor. Cloning by nuclear
transfer makes possible the creation of a near-identical genetic copy of an existing
individual. The closest match can be achieved when the egg and the nucleus come
from the same individual. Even when they do not, only residual mitochondrial DNA
has its origin in the egg, whereas all the other genetic material is derived from the
transferred nucleus.

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How drugs act : Molecular aspects

James M. Ritter DPhil FRCP HonFBPhS FMedSci, in Rang & Dale's Pharmacology,
2020

Cloning of Receptors

In the 1970s, pharmacology entered a new phase when receptors, which had until then
been theoretical entities, began to emerge as biochemical realities following the
development of receptor-labelling techniques (seeCh. 2), which made it possible to
extract and purify the receptor material.

Once receptor proteins were isolated and purified, it was possible to analyse
the amino acid sequence of a short stretch, allowing the corresponding base
sequence of the mRNA to be deduced and full-length DNA to be isolated by
conventional cloning methods, starting from a cDNA library obtained from a tissue
source rich in the receptor of interest. The first receptor clones were obtained in this
way, but subsequently expression cloning and, with the sequencing of the entire
genome of various species, including human, cloning strategies based on sequence
homologies, which do not require prior isolation and purification of the receptor
protein, were widely used, and now several hundred receptors of all four structural
families (seeFig. 3.3) have been cloned. Sequence data so obtained has revealed many
molecular variants (subtypes) of known receptors that had not been evident from
pharmacological studies (see IUPHAR/BPS,Guide to Pharmacology). Much remains
to be discovered about the pharmacological, functional and clinical significance of
this abundant molecular polymorphism. It is expected, however, that such variations
will account for part of the variability between individuals in response to therapeutic
agents (seeCh. 12)

Endogenous ligands for many of the novel receptors identified by gene cloning are so
far unknown, and they are described as ‘orphan receptors’.2 Identifying ligands for
these presumed receptors is often difficult. Increasingly, there are examples (e.g. free
fatty acid receptors) where important endogenous ligands have been linked to hitherto
orphan receptors. There is optimism that novel therapeutic agents will emerge by
targeting this pool of unclaimed receptors.

Much information has been gained by introducing the cloned DNA encoding
individual receptors into cell lines, producing cells that express the foreign receptors
in a functional form. Such engineered cells allow much more precise control of the
expressed receptors than is possible with natural cells or intact tissues, and the
technique is widely used to study the binding and pharmacological characteristics of
cloned receptors. Expressed human receptors, which often differ in their sequence and
pharmacological properties from their animal counterparts, can be studied in this way.

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Cloning
K.H.S. Campbell, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Background and History

The term cloning was originally used to describe the production of genetically
identical copies of an organism by asexual means, for example, the propagation of
plants from cuttings. In animals, the term ‘clone‘ has been applied to offspring
produced by the process of nuclear transfer, whereby a complete and intact genome is
transferred from a donor cell (karyoplast) into a recipient cell which has had its
nuclear DNA removed (cytoplast). The resultant offspring would be a genomic copy
of the nuclear donor; however, in the majority of cases not a true clone as
mitochondrial DNA (mtDNA) present in the recipient cell may differ from that
present in the nuclear donor.

Historically, the technique of nuclear transfer was proposed by Spemann in 1938 as a

method to study differentiation and determine nuclear equivalence. However, due to
technical limitations, successful nuclear transfer was not reported until the 1950s
when Briggs and King reported the production of swimming tadpoles following
transplantation of nuclei from blastula-stage embryos into enucleated frog’s eggs.
Subsequently, John Gurdon reported the generation of adult Xenopus using intestinal
epithelial cells from swimming tadpoles as nuclear donors; however, although
embryos produced using adult keratinocytes as nuclear donors developed into
swimming tadpoles, no adults were obtained. The development of nuclear transfer
techniques in mammals began in the 1970s, and in the early 1980s, successful
development was obtained following the transfer of pronuclei between mouse
zygotes, demonstrating that the manipulation procedures did not prevent subsequent
development. However, the use of enucleated murine zygotes as cytoplast recipients
proved to be extremely restricted when nuclei from later embryonic stages were
transferred. In contrast to the lack of development observed when using enucleated
zygotes as recipients, the use of unfertilized eggs enucleated at metaphase of the
second meiotic division supported the development of live lambs
when blastomeres from 8- to 16-cell embryos were used as nuclear donors.
Subsequently, similar techniques were used in both cattle and pigs.

Although successful, these studies were restricted to the use of embryo-derived

blastomere nuclei which limited application of the technology. Additionally, the
embryonic stage from which successful development could be achieved differed
between species and these differences appeared to reflect a correlation with the onset
of embryonic transcription. This correlation suggested that following the onset of
transcription, the embryonic genome became modified and could not support
development. Despite these limitations, nuclear transfer became recognized in the
agricultural industry as a commercial procedure for producing multiple copies of elite
embryos; however, other factors that affected the efficiencies of embryo and animal
production increased production costs. This coupled with developmental
abnormalities in the offspring restricted commercial application.

To overcome the limitations associated with the use of embryonic blastomeres and
also to address the original questions proposed by Spemann regarding nuclear
equivalence, a major scientific objective in farm animals was to produce offspring
from somatic cells recovered from adult animals. The ability to culture donor cells
prior to use would not only allow production of multiple ‘clones’ but allow storage of
specific genotypes by cryopreservation of the cells and also provide a possible route
to genetic modification. It had been suggested that nuclear transfer from somatic cells
was not possible; however, later studies had shown that inner cell mass cells of sheep
embryos could be used successfully as nuclear donors and it was suggested that
embryonic stem (ES) cells may prove to be suitable nuclear donors. However, to date,
no proven ES cells have been isolated in farm animals. An alternative to isolation of a
suitable cell type was to modify and improve the nuclear transfer technique. Taking
this approach, the first mammals produced by nuclear transfer using cultured
differentiated cells as nuclear donors were born in 1995. Although the cells used as
nuclear donors were derived from an embryo, they developed markers associated with
differentiation during culture. The technique used in these experiments was repeated
using somatic cells derived from an adult animal resulting in the birth of ‘Dolly’ in
1996. Since this time, somatic cell nuclear transfer (SCNT) techniques have been
applied to a wide range of species including mice, cattle, pigs, goats, rats, rabbits,
dogs, cats, horses, mules, deer, wolves, ferrets, and camels as well as rare animals
such as mouflon, ibex, and gaur.

SCNT is a multistage process and numerous technical and biological factors can
affect the success and efficiency of producing healthy animals. These include donor
cell type, recipient cell type, coordination of donor and recipient cell cycle stages,
methods of enucleation fusion and activation, culture of reconstructed embryos, and
preparation and management of surrogate recipients.

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Molecular Biology
Jean L. Bolognia MD, in Dermatology, 2018

The Foundations of Molecular Techniques for Analyzing DNA,

RNA, and Protein

The concepts behind molecular biology are simple and unifying. In general, they
consist of extracting the molecules of interest, amplifying them to measurable
amounts, and detecting them. Polymerase chain reaction (PCR) is a standard
technique for amplifyingDNA (Table 3.1;Fig. 3.4)12. The PCR-amplified DNA,
typically 50 to 2000 base pairs in size depending on the primers designed for a
particular sequence, can be detected in a gel using an intercalating dye that fluoresces
with ultraviolet light. The nucleotide sequence can then be determined via automated
fluorescence sequencing techniques (Table 3.2;Fig. 3.5). This simple and relatively
inexpensive approach is still widely used. However, it is being supplanted by
massively parallel sequencing, also known as next-generation sequencing, in which
millions of fragments of DNA are sequenced in a single run (seeTable 54.6)13.

RNA is also easy to purify, but it is much more readily degraded than DNA.
Therefore, a typical first step in the analysis of RNA is to convert it into DNA
using reverse transcription (RT;Table 3.3;Fig. 3.6A). Following RT, the
complementary DNA (cDNA) can be amplified by PCR, as described above. The
technique of RT-PCR has also been modified to allow accurate quantitation of very
low levels of mRNA14. Because the amount of PCR product is monitored throughout
each cycle of amplification, this technique is referred to as “real-time” quantitative
PCR (Fig. 3.6B).

The amount ofprotein is a complex balance of synthesis and degradation controlled at

multiple steps, including efficiency of protein translation and post-translational
modifications that affect protein stability. One method used to measure levels of
protein is referred to as a Western blot (Table 3.4;Fig. 3.7); it is also known as an
immunoblot because an antibody is employed to detect the protein of interest. In
addition to measuring protein levels, Western blot analysis can determine the size of
proteins and can reveal whether there are different forms of the protein15. Another
method commonly used to measure protein levels is an enzyme-linked
immunosorbent assay (ELISA; seeTable 3.4)16. An ELISA can provide very exact
quantitation of protein levels and may be less expensive and easier to perform than a
Western blot.

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Cloning
Padma Nambisan, in An Introduction to Ethical, Safety and Intellectual Property
Rights Issues in Biotechnology, 2017

2.2.6.7 Others

In New Zealand, research in cloning is pursued by AgResearch Limited which has

an explicit voluntary moratorium ensuring that cloned animals or products do
not enter the food chain. In Japan, the Ministry of Agriculture, Forestry and
Fisheries keeps records of cloned animals and has imposed a voluntary ban on
cloning of livestock except for research purposes (European Commission, 2013).

Key Takeaways
Laws and Public Policy on Reproductive Cloning in Animals:

US Regulated by the CVM of the FDA,

concluded in 2008 that food from clones
safe to eat, released Guidance for
Industry#179 jointly with USDA and
Undersecretary of Marketing and Research
Services; no labeling requirement; no
restriction on sales of clones as
breeding stock
EU 2008—European Group on Ethics find no
ethical justification for cloning
animals, 2013 EC adopts two proposals—
(1) there would be no cloning for farming
purposes in the EU or import of clones,
but research on clones, production of
pharmaceuticals, and endangered species
permitted (2) temporary ban on placing on
the market live clones, embryos of clones
or food from clones
EFSA assessment of food safety in 2008
concluded that food from clones is no
different from that of conventionally
bred livestock
Canada Food from clones defined as novel foods
requiring premarket safety assessment.
Animals for research purposes are
explicitly prohibited from entering food
chain
Argentina Cloning not considered different from
other assisted reproductive techniques
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Cloning
In Stem Cells (Second Edition), 2014

6.2 Cloning Pets: Snuppy, Missy, and Copycat

One application of reproductive cloning considered by some to have commercial

potential is the cloning of pets. Many pet owners, it seems, would like to have an
exact duplicate of their beloved cat or dog, particularly when the animal dies. But how
realistic is this? What would be the cost of cloning a pet? And what would be the
consequences? After the birth of the cloned sheep Dolly it was likely that mammals
such as cats and dogs could be cloned. The American billionaire John Sperling
thought along the same lines and in 1997, together with Bay Area entrepreneur Lou
Hawthorne, decided to clone a dog. They coined the name “Missyplicity” for their
enterprise, after the dog they wanted to clone, Missy, a mixed-breed Border
Collie and Siberian Husky. Sperling and Hawthorne sought scientific expertise in
Texas A&M University and invested approximately US$3.7 million in the
Missyplicity project. However, dog cloning turned out to be much more difficult than
imagined, not least because the ovulated eggs of dogs are not yet ready to be
fertilized, in contrast to ovulated eggs of many other mammals. Instead, these eggs
undergo a large part of meiosis in the oviduct, an environment that is difficult to
recreate in a test tube. Members of the Missyplicity project together with scientists
from Texas A&M University founded Genetic Savings & Clone and diversified their
research to other animals, particularly cats. In 2001 the first cloned cat, aptly named
CopyCat or CC, was born from a calico, or tortoiseshell, donor. Although CC’s donor
was orange, black, and white, CC was black and white and lacked the orange, despite
being genetically identical. The reason for this is that the coat color of cats is
determined epigenetically (Figure 6.9). Outwardly therefore, CC looked completely
different from her twin donor. Although an interesting outcome scientifically,
Hawthorne was disappointed by the result since the commercial success of pet cloning
would largely depend on the pet showing a strong physical resemblance to the
original animal. In 2006 Genetic Savings & Clone ceased to exist; a scientific success
but a commercial failure.
Sign in to download full-size image
Figure 6.9. The beautiful coat pattern of a tortoise-shell cat has its origin in X-chromosome activation. When such a cat is cloned,
her clone will have a different coat pattern since X-chromosome inactivation is a random process.

A new company, called BioArts International Ltd., was set up by Hawthorne,

independent of Texas A&M University. Collaboration was sought this time with the
South Korean Sooam Biotech Research Foundation, with which Woo-Suk Hwang
was associated as part of the research team. Missy had died in 2002, but some of her
cells had been frozen and were flown to the Hwang team in Korea. In December
2007, Mira, the first clone of Missy, was born. Based on this success, an auction was
organized where in a five-day period customers could bid for their dog to be cloned,
but only the five highest bidders would be offered this opportunity. The auction raised
approximately US$750,000 and indicated a potential market for dog cloning. All five
dogs were successfully cloned. However, when BioArts International announced their
Golden Clone Giveaway contest on May 30, 2008, where the prize was a free clone of
the winner’s dog, only 237 people signed up. This low number demonstrated the
extremely limited market for a dog clone on demand, even when it was done for free,
let alone when it would cost tens of thousands of dollars. BioArts International,
therefore, decided to withdraw from dog cloning as a business.

To have your dog cloned, South Korea is still the place to be, however. South Korean
Byeong-Chun Lee from Seoul National University, a former colleague of Hwang and
part of the “Snuppy team,” now works with the company RNL Bio. He has cloned a
series of dogs, including a drug-sniffing dog because it was so outstanding and unique
in its job. In 2008, the team presented its first commercially cloned Pitbull Terrier,
“Booger,” which had cost the owner an estimated US$50,000.
Despite the limited market, Sooam BRF announced on 22 March 2013 the first dog
cloning competition for the United Kingdom. Just as the giveaway contest of 2008,
applicants could send in a reason why their dog should be cloned. This time, however,
the competition was restricted to residents of the United Kingdom. The winner has
been promised that his or her dog will be cloned for free, provided that the winner
agrees to participate in a documentary about the process.

Aside from any commercial application, dog cloning could help in providing new
scientific knowledge. Several dog breeds suffer diseases similar to those in humans,
and these dogs might be useful as animal models for human conditions. The
availability of genetically identical dogs would eliminate the confounding factor
of genetic diversity between individual animals in the disease studies. Moreover, dogs
can be valuable for studying brain function and behavior, for instance. Again, cloning
groups of dogs could circumvent the common problem of genetic variation normally
present within dog groups. Pet cloning is controversial, however. Not only because
dog cloning at Sooam is led by the once-disgraced Dr. Hwang, but, more importantly,
because the costs are enormous, while pet shelters continue to become fuller each
year.

6.4
The Cloning of Dolly
Interview with Ian Wilmut
How did you come to clone a sheep?
Our objective was to be able to improve the health and productivity of farm animals.
Cattle are by far the most important of farm animals, but they are extremely
expensive. We had a great deal of experience of recovering and culturing sheep
embryos and knew that embryo development in sheep is very similar to that in cattle.
So we chose to work with sheep because they are cheap and we were confident that
methods developed in sheep would be readily adaptable for the cow. That has, in fact,
proved to be the case.
How did you feel when you realized Dolly’s biological mother was pregnant?
Of course, we were very excited when we discovered that Dolly’s mother was
pregnant. She was approximately six weeks into pregnancy when ultrasound scanning
first showed that she was carrying a lamb. However, previous experience had shown
us that a considerable proportion of the fetuses that were present at that stage of
development died during pregnancy or at the time of birth. So we were extremely
cautious in our expectations from the time of the first scan until a few weeks after her
birth when we became confident that Dolly was healthy and viable.
What did the birth of Dolly change in science?
The birth of Dolly provided important new understanding of the mechanisms that
regulate mammalian development. Her birth showed that the mammary cell used in
the cloning process contained all of the genetic information necessary to produce a
viable offspring. Earlier, researchers had suggested that cells formed different tissues
by losing segments of the chromosomal DNA that were not required for the
functioning of a particular tissue. Clearly this was not the case (Figure B6.4.1).
Sign in to download full-size image
Figure B6.4.1. Sir Ian Wilmut and Dolly.
Source: Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh,
Edinburgh.

The current hypothesis is that differentiation to form all the tissues of an adult is
brought about by a sequence of changes in gene expression, which become
progressively more specific for the final tissue. Before the birth of Dolly it was
believed that the mechanisms that regulate these changes are so complex and so
rigidly fixed that they cannot be reversed by the process of nuclear transfer. It was
suggested that this was the reason why it had not previously been possible to produce
a clone from an adult animal. The birth of Dolly clearly showed that this was not the
case and has led to very important research to find methods that enable us to produce
cells that closely resemble embryo stem cell from adult tissue. These so-called
“induced pluripotent cells” will have a very profound effect in biomedical research.
They are the most important outcome from the Dolly experiment.
What impact did Dolly have on you your life and career?
The project was led by Keith Campbell and I, and the birth of Dolly transformed our
lives. We have both had greater opportunities to develop our careers following the
birth of Dolly. Keith became a professor at the University of Nottingham and I
became director of a research center in the University of Edinburgh. The other effect
of the experiment is that we have both given an enormous number of interviews to
radio, television, and newspapers.