Describes different types of cloning, advantages and disadvantages of cloning, ethical implications of cloning animals etc.

1. Cloning
Dr. Radhakrishna G Pillai
(MSc (KU), MSc (London), Ph.D, LLB, BEd, PGCE (London), SLTP (London), PGDDE)

2. What is cloning in biology
• Variable meaning in biology
• Obtaining copies more or less precise of a biological entity
• Three common types of cloning;
– cloning genes
– cloning cells and
– cloning individuals
• Cloning an individual: in case of a multicellular organism, such as a
plant or an animal is not strictly possible
• The genes of an individual, the genome, can be cloned, but
• The individual itself cannot be cloned

3. Cellular Cloning
• Unicellular organisms such as;
– bacteria and yeast
– naturally produce clones of themselves
– During their asexual reproduction
• Known as cellular cloning
• The nuclear DNA duplicates by the process of
mitosis, which creates an exact replica of the
genetic material

4. Different forms of
vegetative reproduction
in microbes

5. Human cloning
• Suggested as a way to improve the genetic endowment of
mankind:
– by cloning individuals of great achievement
– for example, in sports, music, the arts, science, literature,
politics, and the like, or of acknowledged virtue
• The clone may not be exact copy phenotypically: never
been taken seriously
• The obstacles and drawbacks are many and insuperable, at
least at the present state of knowledge
• Many advertise that they were ready to carry out the
cloning

6. Cloning genes/cell
• Cloning genes or, more generally,
cloning DNA segments is routinely
done in many genetics and
pharmaceutical laboratories
throughout the world
• Technologies for cloning cells in the
laboratory are seven decades old
and
• Cloning of cells is used for
reproducing a particular type of
cell
• Eg. skin or liver cell: to investigate
its characteristics

7. Natural cloning
• Individual human cloning occurs naturally in the
case of identical twins
• Two individuals develop from a single fertilized egg
• These twins are called identical, precisely because
they are genetically identical to each other
• The sheep Dolly, cloned in July 1996
– the first mammal artificially cloned using an adult
cell as the source of the genotype
• Frogs and other amphibians were obtained by
artificial cloning as early as 50 y earlier

8. Can a human individual be cloned?
• Strictly speaking: No
• What is cloned are the genes, not the
individual; the genotype, not the phenotype
• The technical obstacles are immense even for
cloning a human’s genotype

9. Success rate
• Ian Wilmut Succeeded with Dolly only
after 270 trials
• The rate of success for cloning
mammals has notably increased over
the years but far below 100%
• Presently mice, rats, goats, sheep,
cows, pigs, horses, and other
mammals are cloned
• The great majority of pregnancies end
in spontaneous abortion
• In many cases, the death of the fetus
occurs close to term, with devastating
economic, health, and emotional
consequences

10. Reproductive cloning
• The deliberate production of genetically
identical individuals
• Each newly produced individual is a clone of
the original
• Clones contain identical sets of genetic
material in their chromosomes—of every cell
in their bodies
• Cells from two clones have the same DNA and
the same genes in their nuclei

11. Mitochondrial DNA
• Cells also contain some DNA in the
mitochondria
• Mitochondria contain their own DNA
and reproduce independently
• True clones will have identical DNA in
both the nuclei and mitochondria
• Individuals that have identical
nuclear DNA but different
mitochondrial DNA is also called
clones

12. Cloning using somatic cell nuclear
transfer (SCNT)
• Remove the chromosomes (Nucleus) from an
egg: enucleated egg
• The chromosomes are replaced with a nucleus
from a somatic (body) cell
• Somatic cell from the donor of egg cell or from
another individual
• Cells obtained directly from the individual, cells
grown in culture, or frozen tissue
• Transfer to the uterus of synchronized recipient
mothers
• Carried to term to produce live cloned off-spring

13. SCNT
• The egg is then stimulated and in some cases it starts to divide
• If that happens, a series of sequential cell divisions leads to
the formation of a blastocyst, or preimplantation embryo
• The blastocyst is then transferred to the uterus of an animal
• The successful implantation of the blastocyst result in its
further development, culminating in the birth of an animal
• This animal will be a clone of the individual that was the
donor of the nucleus
• Its nuclear DNA has been inherited from only one genetic
parent

14. Limitations in SCNT
• The number of times that a given individual can be cloned is
limited by the number of eggs that can be obtained
• The number of females available to receive developing embryos
• Approximately 40-50% of the reconstructed embryos develop to a
stage suitable for transfer
• Only about 10% result in the birth of a live cloned calf
• Currently only about 4-5% of cloned embryos originally
constructed actually make it to become calves

15. Inheritance of DNA
• If the egg and somatic cell are from the same individual:
– result an embryo that receives all its genetic material—nuclear
and mitochondrial—from a single individual
• That will also be true if the egg comes from the nucleus
donor's mother: mitochondria are inherited maternally
• Multiple clones might also be produced by transferring
identical nuclei to eggs from a single donor
• If the somatic cell nucleus and the egg come from different
individuals: clone not identical to the nuclear donor as
mitochondrial genes are different

16. Cloning by embryo splitting
• Begins with in vitro fertilization (IVF)
• The zygote divides into two and then
four identical cells
• Separate the cells and allowed to
develop into separate but identical
blastocysts
• Implant the blastocyst in a uterus
• Limited developmental potential
• Embryo splitting can yield only two
identical and probably no more than
four identical individuals

17. Similarity in DNA
• The DNA in embryo splitting is contributed by
germ cells from two individuals
• Thus, the embryos, like those formed naturally
or by standard IVF, have two parents
• Their mitochondrial DNA is identical
• This method of cloning is identical with the
natural formation of monozygotic twins

18. WILL CLONES LOOK AND BEHAVE THE SAME?
• Clones are genetically identical with one another
• They will not be identical in physical or behavioral
characteristics
• DNA is not the only determinant of these
characteristics
• A pair of clones will experience different environments
and nutritional inputs while in the uterus,
• Subjected to different inputs from their parents,
society, and life experience as they grow up

19. Differences in phenotype of clones
• Clones with identical nuclear and identical
mitochondrial DNA
– but born at different times
– the environmental and nutritional differences
– would be expected to be more pronounced than for
monozygotic (identical) twins
• Monozygotic twins may not be fully identical
genetically or epigenetically:
– mutations, stochastic developmental variations, and
varied imprinting effects (parent-specific chemical marks
on the DNA) make different contributions to each twin

20. Mitochondrial DNA
• Clones that do not have identical mitochondria are
genetically different
• Such clones arise if one individual contributes the
nucleus and another the egg
• Nuclei from a single individual transferred to eggs from
multiple donors are also genetically different
• The differences in mitochondrial DNA might be
expected to show up in parts of the body that have
high demands for energy
– such as muscle, heart, eye, and brain or
– in body systems that use mitochondrial control over cell
death to determine cell numbers

21. Why Reproductive cloning?
• Cloning of livestock is a means of
– replicating an existing favorable combination of traits, such as efficient
growth and high milk production
– exclude the impact of genetic “lottery” and
– mixing that occur in sexual reproduction
• It allows an animal with a particular genetic modification, such as
the ability to produce a pharmaceutical in milk, to be replicated
more rapidly than does natural mating
• Moreover a genetic modification can be made more easily
– genetic modifications in cultured cells and
– the modified cell nucleus transferred to an enucleated egg to make a
clone
• Mammals used in scientific experiments, such as mice, are cloned as
part of research
– To increase our understanding of fundamental biological mechanisms

22. Human reproductive cloning
• People wish to produce children through
reproductive cloning include:
– Individuals who wish to have a child that is genetically
identical with them, or with another nucleus donor
• Infertile couples
– Parents who have lost a child and wish to have another
genetically identical child
– People who need a transplant (Eg. cord blood) to
• treat their own or their child's disease and
• collect genetically identical tissue from a cloned foetus or
newborn

23. Reproductive cloning
• Transplantable tissue may be available without
the need for the birth of a child produced by
cloning
• For example, embryos produced by in
vitro fertilization (IVF) can be typed for transplant
suitability
• In future stem cells produced by nuclear
transplantation may allow the production of
transplantable tissue

24. Stem cells through SCNT
• Somatic cell nuclear transfer or nuclear transplantation (NT):
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 non-reproductive cloning
– would be used to generate pluripotent ES cells
– that are genetically identical with the cells of a transplant recipient
• Avoid/ameliorate the rejection seen with unmatched transplants
• The ES cells from NT have the advantage over adult stem cells
– being able to provide virtually all cell types and
– able to be maintained in culture for long periods of time
• In the future they might be used to generate tissues and,
theoretically, complex organs for transplantation

25. Stem cell therapy through NT
• Transfer of a somatic cell nucleus
from a patient into an enucleated
egg
• in vitro culture of the embryo to the
blastocyst stage and
• Pluripotent ES cell line from the inner
cell mass of this blastocyst
• Derive specialized cells (and, if
possible, tissues and organs) in
laboratory culture for therapeutic
transplantation

26. Stem cells from SCNT
• Could avoid a major cause of
transplant rejection
Possible drawbacks of this
• Presence of divergent
mitochondrial proteins in cells
may create “minor”
transplantation antigens
– that can cause rejection
• Not be a problem: if the egg were
donated by the mother of the
transplant recipient or the
recipient herself

27. Autoimmune diseases
• For some autoimmune diseases, transplantation of cells
cloned from the patient's own cells may be inappropriate
• These cells can be targets for the ongoing destructive process
• In disorder that has a genetic origin: ES cells derived by SCNT
from the patient's own cells would carry the same defect
– Need genetic modification before they could be used for
therapeutic transplantation
• Using another source of stem cells is more likely to be
feasible 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

28. Uses of stem cells from SCNT
• Therapeutic use
• Used in laboratories for several types of studies in clinical medicine
and for fundamental research in human development
• 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
– Eg. ES cells derived from humans with genetic
– permit analysis of the role of the mutated genes in both cell and tissue
development and in adult cells difficult to study otherwise.
– Eg nerve cells of the brain
• Disadvantage: 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
– the derivation of primary cell lines from human tissues is not possible

29. Artificial Embryo Twinning
• Artificial embryo twinning is a relatively low-tech
way to make clones
• This mimics the natural process that creates
identical twins
• In nature, twins form very early in development
when the embryo splits in two
• Twinning happens in the first days after egg and
sperm join
– the embryo is made of just a small number of
unspecialized cells
• Each half of the embryo continues dividing on its
own, ultimately developing into separate,
complete individuals
• From the same fertilized egg: genetically identical

30. Artificial embryo twinning
• Uses the same approach as natural embryo
twinning
• It is carried out in a Petri dish instead of
inside the mother
• A very early embryo is separated into
individual cells, which are allowed to divide
and develop for a short time in the Petri dish
• The embryos are then placed into a
surrogate mother, where they finish
developing
• Since all the embryos came from the same
fertilized egg, they are genetically identical

31. https://www.scientificamerican.com/article/20-years-after-dolly-the-sheep-led-the-way-
where-is-cloning-now/

32. Issues to be resolved
• Birth of Dolly happened only after 270 trials
• The great majority of pregnancies end in spontaneous
abortion
• The death of the fetus occurs close to term
– with devastating economic, health, and emotional consequences in
the case of humans
• In mammals, in general, the animals produced by cloning
suffer from serious health handicaps
– gross obesity
– early death
– distorted limbs and
– dysfunctional immune systems and organs (including liver and
kidneys) &
– other mishaps

33. Can we clone an individual, Why?
• It might be possible to clone a person’s genes
• But the individual cannot be cloned
• The character, personality, and the features
other than anatomical and physiological that
make up the individual are not precisely
determined by the genotype

34. Dolly… Why not primates?
• The cloning technique used to create Dolly has been
shown not to work in primates
• likelihood is pregnancy losses, abnormal births
• one of the lambs cloned in the same lab soon after
Dolly developed lung problems that caused it to
hyperventilate and regularly pass out
• Recent advances in gene-editing technology, the need
for cloning to correct genetic errors will decline even
further

35. Benefits
• Three main applications for agriculture
– the rapid spread of animals with better genetic
characteristics
– Increased availability of stocks with desirable traints
– Controlled characters
• Theoretically, cloning could also be used to bring
back endangered species
• Somatic cell nuclear transfer may help researchers
better understand early human embryogenesis
and stem cell biology

36. Cloning benefits
• Cloning solve some of the problems that the dairy
industry currently faces in the form of decreased fertility
• Rapidly increase the number, distribution, and availability
of cows and bulls with superior genetics that could allow
increases in milk yield
• Increases in the availability of stock with
– resistance to common diseases such as mastitis, and
– desirable traits associated with milk quality
• The further development of transgenics could
– enhance these functions and also
– increase the ability to produce specialized bioproducts for use
in medicine, pharmaceuticals, and many other industries.
• More difficult to quantify and measure at this early stage of
development

37. Problems associated with cloning
• Large number of technological difficulties and problems to overcome
• Use of cloning technologies are delayed due to;
– current success rates
– variability of results
– costs
– government regulation and
– public perception and acceptance
• In January 2008, the US Food and Drug Administration released a
comprehensive study that concluded that meat and milk from cloned
animals is safe for consumers
• There is still wide-spread uncertainty and significant debate about the
feasibility and acceptability of animal cloning as a technology
• Cloning is commercially available, but the technology still is considered to
be quite inefficient and very costly

38. Extended gestation
• Gestation is usually extended and calves are born much larger
than average
• Due to large offspring syndrome (LOS)
• That leads to dystocia and most animals require cesarean
section
• These large offspring usually have postnatal weakness;
– hypoxia, hypoglycemia, metabolic acidosis, and hypothermia,
all requiring immediate intensive care
• Postnatal problems could be compounded from one
generation to the next
• Cloning will reduce genetic diversity and result in
problems of fertility and disease susceptibility

39. Inbreeding and loss of genetic variation
• Unlimited numbers of identical animals could be produced with
cloning
• Overpopulation of the same genetic makeup could result in inbreeding
and loss of genetic variation, which is not desirable
• With careful management and planning of breeding schemes, these
problems should not occur
• When used correctly, cloning of selected livestock potentially offers
many advantages in animal breeding and production:
– One of which is to preserve biodiversity
• If done without limitations;
– also has the potential to reduce genetic variability
– even in large dairy cattle populations without cloning, a substantial
reduction in genetic variability is already occurring
– because of the massive use of relatively small numbers of sires which are
quite often genetically related

40. Can we overcome?
• New technologies could help to overcome such problems
• Preimplantation genetic diagnosis (PGD)
– reducing the costs of some of the problems associated with
cloning and other advanced breeding technologies
– Embryos can be pre-screened, thus allowing the elimination
of genetically abnormal embryos
– Same technique used to select embryos with economically
beneficial traits
• Clones may be phenotypically similar in simply inherited
characteristics (identical in color, shape, size, etc.)

41. Similarity in cloned individuals
• Gnetic performance may not be exactly the same as the
clonal parents
• Some similarity depending on the heritability of the
characteristics
• In most cases there is no certainty that a clone will be superior,
and in many cases, the clone may actually be inferior
• Cloning could provide faster dissemination of superior genetic
material to the population than previous breeding schemes
have achieved
• but costs are currently extremely high and
• Legal, ethical, and economic questions remain

42. Ethical aspects
• UNESCO's Universal Declaration on the Human Genome and Human
Rights asserts that the reproductive cloning of human beings is
contrary to human dignity
• A potential life represented by the embryo is destroyed when
embryonic cells are used
• The inherent unreliability of cloning technology would be damaging
to cloned individuals
• Non-human animals also possess certain moral rights as living
entities and should therefore be afforded the same ethical
considerations as human beings
• Stand against the exploitation of animals in scientific research on
cloning, cloning for food production, or other uses like therapeutic
purposes etc

43. Different difficulties of human clones
– Human cloning might change the shape of family structure by
complicating the role of parenting within a family of convoluted
kinship relations
• Eg. a female DNA donor would be the clone's genetic twin rather than
mother
– These facts complicate the genetic and social relationships between
mother and child
– As well as the relationships between other family members and the
clone
– Clone would act identically to the human from which they were
cloned: legal, ethical and moral conflicts
– This could infringe on the right to self-determination

44. Cloning: moral issues
• Cloned animals are used in scientific and medical research such
as;
– therapeutic cloning, stem cell research and human antibody production
• It can be painful for the animal and often result in mental and
physical damage
• Clones created for medical purpose have very poor quality of
life as research procedures are constantly being conducted on
them
• Pet cloning: overwhelming numbers of unwanted companion
animals
• Companies that offer pet cloning are deceiving and exploiting
grieving pet owners