How Is Cloning Done?

Many people first heard of cloning when Dolly the Sheep showed up on the scene in 1997. Artificial cloning
technologies have been around for much longer than Dolly, though.

There are two ways to make an exact genetic copy of an organism in a lab: artificial embryo twinning and somatic
cell nuclear transfer.

1. Artificial Embryo Twinning

Artificial embryo twinning is a relatively low-tech way to make clones. As the name suggests, this technique 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, while 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. Since they
developed from the same fertilized egg, the resulting individuals are genetically identical.

Artificial embryo twinning uses the same approach, but 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. Again, since all
the embryos came from the same fertilized egg, they are genetically identical.

2. Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT), also called nuclear transfer, uses a different approach than artificial embryo
twinning, but it produces the same result: an exact genetic copy, or clone, of an individual. This was the method
used to create Dolly the Sheep.

What does SCNT mean? Let's take it apart:

Somatic cell: A somatic cell is any cell in the body other than sperm and egg, the two types of reproductive cells.
Reproductive cells are also called germ cells. In mammals, every somatic cell has two complete sets of
chromosomes, whereas the germ cells have only one complete set.

Nuclear: The nucleus is a compartment that holds the cell's DNA. The DNA is divided into packages called
chromosomes, and it contains all the information needed to form an organism. It's small differences in our DNA
that make each of us unique.

Transfer: Moving an object from one place to another. To make Dolly, researchers isolated a somatic cell from an
adult female sheep. Next they removed the nucleus and all of its DNA from an egg cell. Then
they transferred the nucleus from the somatic cell to the egg cell. After a couple of chemical tweaks, the egg cell,
with its new nucleus, was behaving just like a freshly fertilized egg. It developed into an embryo, which was
implanted into a surrogate mother and carried to term. (The transfer step is most often done using an electrical
current to fuse the membranes of the egg and the somatic cell.)

The lamb, Dolly, was an exact genetic replica of the adult female sheep that donated the somatic cell. She was the
first-ever mammal to be cloned from an adult somatic cell.

How does SCNT differ from the natural way of making an embryo?

Natural fertilization, where egg and sperm join, and SCNT both make the same thing: a dividing ball of cells, called
an embryo. So what exactly is the difference between the two?

An embryo's cells all have two complete sets of chromosomes. The difference between fertilization and SCNT lies in
where those two sets come from.

In fertilization, the sperm and egg have one set of chromosomes each. When the sperm and egg join, they grow
into an embryo with two sets—one from the father's sperm and one from the mother's egg.

In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the nucleus from a somatic cell,
which already contains two complete sets of chromosomes. So, in the resulting embryo, both sets of chromosomes
come from the somatic cell.

Is cloning an organism the same as cloning a gene?

You may have heard about researchers cloning, or identifying, genes that are responsible for various medical
conditions or traits. What's the difference?
When scientists clone an organism, they are making an exact genetic copy of the whole organism, as described
above.

When scientists clone a gene, they isolate and make exact copies of just one of an organism's genes. Cloning a gene
usually involves copying the DNA sequence of that gene into a smaller, more easily manipulated piece of DNA, such
as a plasmid. This process makes it easier to study the function of the individual gene in the laboratory.

The History of Cloning

Lost in the midst of all the buzz about cloning is the fact that cloning is nothing new: its rich scientific
history spans more than 100 years. The landmark examples below will take you on a journey through time,
where you can learn more about the history of cloning.
1885 - First-ever demonstration of artificial embryo twinning
Sea urchin
Hans Adolf Eduard Driesch

The sea urchin is a relatively simple organism that is useful for studying development. Dreisch showed that
by merely shaking two-celled sea urchin embryos, it was possible to separate the cells. Once separated,
each cell grew into a complete sea urchin.

This experiment showed that each cell in the early embryo has its own complete set of genetic instructions
and can grow into a full organism.

1902 - Artificial embryo twinning in a vertebrate

Salamander
Hans Spemann
Spemann’s first challenge was to figure out how to split the two cells of an embryo much stickier than sea
urchin cells. Spemann fashioned a tiny noose from a strand of baby hair and tightened it between two cells
of a salamander embryo until they separated. Each cell grew into an adult salamander. Spemann also tried
to divide more advanced salamander embryos using this method, but he found that cells from these
embryos weren’t as successful at developing into adult salamanders.

This experiment showed that embryos from a more-complex animal can also be “twinned” to form multiple
identical organisms—but only up to a certain stage in development.

1928 - The cell nucleus controls embryonic development

Salamander
Hans Spemann

Again using a strand of baby hair tied into a noose, Spemann temporarily squeezed a fertilized salamander
egg to push the nucleus to one side of the cytoplasm. The egg divided into cells—but only on the side with
the nucleus. After four cell divisions, which made 16 cells, Spemann loosened the noose, letting the
nucleus from one of the cells slide back into the non-dividing side of the egg. He used the noose to separate
this “new” cell from the rest of the embryo. The single cell grew into a new salamander embryo, as did the
remaining cells that were separated.

Essentially the first instance of nuclear transfer, this experiment showed that the nucleus from an early
embryonic cell directs the complete growth of a salamander, effectively substituting for the nucleus in a
fertilized egg.

1952 - First successful nuclear transfer

Frog
Robert Briggs and Thomas King

Briggs and King transferred the nucleus from an early tadpole embryo into an enucleated frog egg (a frog
egg from which the nucleus had been removed). The resulting cell developed into a tadpole.

The scientists created many normal tadpole clones using nuclei from early embryos. But just like
Spemann’s salamander experiments, cloning was less successful with donor nuclei from more advanced
embryos: the few tadpole clones that did survive grew abnormally.
Most importantly, this experiment showed that nuclear transfer was a viable cloning technique. It also
reinforced two earlier observations. First, the nucleus directs cell growth and, ultimately, an organism’s
development. Second, embryonic cells early in development are better for cloning than cells at later stages.

1958 - Nuclear transfer from a differentiated cell

Frog
John Gurdon

Gurdon transplanted the nucleus of a tadpole intestinal cell into an enucleated frog egg. In this way, he
created tadpoles that were genetically identical to the one from which the intestinal cell was taken.

This experiment showed that, despite previous failures, nuclei from somatic cells in a fully developed
animal could be used for cloning. Importantly, it suggested that cells retain all of their genetic material
even as they divide and differentiate (although some wondered if the donor DNA came from a stem cell,
which can differentiate into multiple types of cells).

1975 - First mammalian embryo created by nuclear transfer

Rabbit
J. Derek Bromhall

Mammalian egg cells are much smaller than those of frogs or salamanders, so they are harder to
manipulate. Using a glass pipette as a tiny straw, Bromhall transferred the nucleus from a rabbit embryo
cell into an enucleated rabbit egg cell. He considered the procedure a success when a morula, or advanced
embryo, developed after a couple of days.

This experiment showed that mammalian embryos could be created by nuclear transfer. To show that the
embryos could continue developing, Bromhall would have had to place them into a mother rabbit's womb.
He never did this experiment.
1984 - First mammal created by nuclear transfer
Sheep
Steen Willadsen

Willadsen used a chemical process to separated one cell from an 8-cell lamb embryo. The he used a small
electrical shock to fuse it to an enucleated egg cell. As luck would have it, the new cell started dividing.

By this time, in vitro fertilization techniques had been developed, and they had been used successfully to
help couples have babies. So after a few days, Willadsen placed the lamb embryos into the womb of
surrogate mother sheep. The result was the birth of three live lambs.

This experiment showed that it was possible to clone a mammal by nuclear transfer—and that the clone
could fully develop. Even though the donor nuclei came from early embryonic cells, the experiment was
considered a great success.

1987 - Nuclear transfer from embryonic cell

Cow
Neal First, Randal Prather, and Willard Eyestone

Using methods very similar to those used by Willadsen on sheep, First, Prather, and Eyestone produced
two cloned calves. Their names were Fusion and Copy.

This experiment added cows to the list of mammals that could be cloned by nuclear transfer. Still,
mammalian cloning was limited to using embryonic cells as nuclear donors. Cloning using nuclei from
differentiated adult somatic cells still wasn’t thought possible.
1996 - Nuclear transfer from laboratory cells
Sheep
Ian Wilmut and Keith Campbell

All previous cloning experiments used donor nuclei from cells in early embryos. In this experiment, the
donor nuclei came from a slightly different source: cultured sheep cells, which were kept alive in the
laboratory.

Wilmut and Campbell transferred the nuclei from cultured cells into enucleated sheep egg cells. The lambs
born from this procedure were named Megan and Morag.

This experiment showed that cultured cells can supply donor nuclei for cloning by nuclear transfer.
Because scientists had already learned how to transfer genes into cultured cells, this experiment showed
that it might be possible to use such modified cells to create transgenic animals—such as cows that could
make insulin for diabetics in their milk.

1996 - Dolly: First mammal created by somatic cell nuclear

transfer
Sheep
Ian Wilmut and Keith Campbell

In this landmark experiment, Wilmut and Campbell created a lamb by transferring the nucleus from an
adult sheep's udder cell into an enucleated egg. Never before had a mammal been cloned from an adult
somatic cell. What was the big deal?

Every cell’s nucleus contains a complete set of genetic information. However, while embryonic cells are
ready to activate any gene, differentiated adult cells have shut down the genes that they don't need for their
specific functions. When an adult cell nucleus is used as a donor, its genetic information must be reset to an
embryonic state. Often the resetting process is incomplete, and the embryos fail to develop.

Of 277 attempts, only one produced an embryo that was carried to term in a surrogate mother. This famous
lamb, named Dolly, brought cloning into the limelight. Her arrival started conversations about the
implications of cloning, bringing controversies over human cloning and stem cell research into the public
eye.

1997 - First primate created by embryonic cell nuclear

transfer
Rhesus monkey
Li Meng, John Ely, Richard Stouffer, and Don Wolf

Primates are good models for studying human disorders. Cloning identical primates would decrease the
genetic variation of research animals, and therefore the number of animals need in research studies.

Similar to previous cloning experiments, Wolf’s team of scientists fused early-stage embryonic cells with
enucleated monkey egg cells using a small electrical shock. The resulting embryos were then implanted
into surrogate mothers. Out of 29 cloned embryos, two monkeys were born. One was a female named Neti,
and the other was a male named Ditto.

This experiment showed that primates, humans’ closest relatives, can be cloned.

1997 - Nuclear transfer from genetically engineered

laboratory cells
Sheep
Angelika Schnieke, Keith Campbell, Ian Wilmut

This experiment was an exciting combination of findings from earlier work. Campbell and Wilmut had
already created a clone using the nucleus of a cultured cell. This time, the researchers introduced the human
Factor IX (“factor nine”) gene into the genome of sheep skin cells grown in a laboratory dish. Factor IX
codes for a protein that helps blood clot, and it's used to treat hemophilia, a genetic disorder where blood
doesn't form proper clots.

To create the transgenic sheep, the scientists performed nuclear transfer using donor DNA from the
cultured transgenic cells. The result was Polly, a sheep that produced Factor IX protein in her milk.
This experiment showed that sheep could be engineered to make therapeutic and other useful proteins in
their milk, highlighting the potential medical and commercial uses for cloning.

1998-1999 - More mammals cloned by somatic cell nuclear

transfer
Mice, cows, and goats
Multiple groups

After the successes leading up to Dolly and Polly, other scientists wanted to see if similar techniques could
be used to clone other mammalian species. Before long, several more animals had been successfully
cloned. Among them were transgenic animals, clones made from fetal and adult cells, and a male mouse;
all previous clones had been female.

2001 - Endangered animals cloned by somatic cell nuclear

transfer
Gaur and Mouflon
Multiple groups

As the list of successfully cloned animals grew, scientists began to explore cloning as a way to create
animals belonging to endangered or extinct species. A challenge to cloning endangered and extinct species
is finding closely related animals to serve as egg donors and surrogates. The gaur and mouflon were chosen
in part because they are close relatives of domestic cattle and sheep, respectively.

In 2009, using goast as egg donors and surrogates, another group of researchers cloned the first extinct
animal, a Spanish mountain goat called the bucardo. Sadly, the one kid that survived gestation died soon
after birth due to a lung defect.

2007 - Primate embryonic stem cells created by somatic cell

nuclear transfer
Rhesus monkey
Shoukhrat Mitalipov and colleagues

Researchers took a cell from an adult monkey and fused it with an enucleated egg cell. The embryo was
allowed to develop for a time, then its cells were grown in a culture dish. These cells, because they can
differentiate to form any cell type, are called embryonic stem cells.

This experiment showed that nuclear transfer in a primate, which researchers had tried for years without
success, was possible. It opened the door to the possibility of human therapeutic cloning: creating
individual-specific stem cells that could be used to treat or study diseases.

2013 - Human embryonic stem cells created by somatic cell

nuclear transfer
Human
Shoukhrat Mitalipov and colleagues

Overcoming decades of technical challenges, Mitalipov and colleagues were the first to use somatic cell
nuclear transfer to create a human embryo that could be used as a source of embryonic stem cells. The
resulting stem cell lines were specific to the patient they came from, a baby with a rare genetic disorder.

In this experiment, researchers took a skin cell from the patient and fused it with a donated egg cell. Key to
the success of the experiment were modifications to the culture liquid in which the procedure was done and
to the series of electrical pulses used to stimulate the egg to begin dividing.

Following the cloning controversy of 2004–2005, in which South Korean scientists falsely claimed to have
used somatic cell nuclear transfer to create embryonic stem cell lines, the scientific community demanded
much stronger evidence that the procedure had actually been successful.
Why Clone?
Our experiences have told us that, with a little work, we humans can clone just about anything we want,
from frogs to sheep—and probably even ourselves.

So we can clone things. But why would we want to? Below are some of the ways in which cloning might
be useful.

Cloning in Medicine
Cloning for medical purposes has the potential to benefit large numbers of people. How might cloning be
used in medicine?

Cloning animal models of disease

Much of what researchers learn about human disease comes from studying animal models such as mice.
Often, animal models are genetically engineered to carry disease-causing mutations in their genes. Creating
these transgenic animals is a time-intensive process that requires trial-and-error and several generations of
breeding. Cloning could help reduce the time needed to make a transgenic animal model, and the result
would be a population of genetically identical animals for study.

Cloning to make stem cells

Stem cells build, maintain, and repair the body throughout our lives. Because these are processes that stem
cells do naturally, they can be manipulated to repair damaged or diseased organs and tissues. But stem cells
transferred from one person to another (such as in a bone marrow transplant) are seen as foreign, and they
usually trigger an immune response.

Some researchers are looking at cloning as a way to create stem cells that are genetically identical to an
individual. These cells could then be used for medical purposes, possibly even for growing whole organs.
And stem cells cloned from someone with a disease could be grown in culture and studied to help
researchers understand the disease and develop treatments.

In 2013, scientists at Oregon Health and Science University were the first to use cloning techniques to
successfully create human embryonicstem cells. The donor DNA came from an 8-month-old with a rare
genetic disease.

Find out more about Stem Cells.

 Reviving Endangered or Extinct Species
You might have seen the Jurassic Park movies. In the original feature film, based on the Michael Crichton
novel, scientists use DNA preserved for tens of millions of years to clone dinosaurs. They run into trouble,
however, when they realize that the cloned creatures were smarter and fiercer than expected. Could we
really clone dinosaurs?

In theory? Yes. You would need:

 A well-preserved source of DNA from the extinct dinosaur, and

 A closely related species, currently living, that could serve as an egg donor and surrogate mother.

In reality? Probably not.

It's extremely unlikely that dinosaur DNA could survive undamaged for such a long time. However,
scientists have been working to clone species that became extinct more recently, using DNA from well-
preserved tissue samples. A number of projects are underway to clone extinct species, including the wooly
mammoth.

In 2009, scientists had their first near-success resurrecting an extinct animal. Using goats as egg donors and
surrogates, they made several clones of a wild mountain goat called the bucardo—but the longest-surviving
clone died soon after birth. Even if the effort eventually succeeds, the only frozen tissue sample comes
from a female, so it will only produce female clones. However, scientists speculate they may be able to
remove one X chromosome and add a Y chromosome from a related goat species to make a male.

Cloning endangered species is much easier, mainly because the surviving animals can donate healthy,
living cells. In fact, several wild species have been cloned already, including two relatives of cattle called
the guar and the banteng, mouflon sheep, deer, bison, and coyotes. However, some experts are skeptical
that cloning can help a species recover. One big challenge endangered species face is the loss of genetic
diversity, and cloning does nothing to address this problem. When a species has high genetic diversity,
there is a better chance that some individuals would have genetic variations that could help them survive an
environmental challenge such as an infectious disease. Cloning also does not address the problems that put
the species in danger in the first place, such as habitat destruction and hunting. But cloning may be one
more tool that conservation scientists can add to their toolbox.
Reproducing a Deceased Pet
If you really wanted to, and if you had enough money, you could clone your beloved family cat. At least
one biotechnology company in the United States has offered cat cloning services for the privileged and
bereaved. But don't assume that your cloned kitty will be exactly the same as the one you know and love.
An individual is a product of more than its genes—the environment plays an important role in shaping
personality and many other traits.

On December 22, 2001, a kitten named CC made history as the first cat—and the first domestic pet—ever
to be cloned. CC and Rainbow, the donor of CC's genetic material, are pictured at the right.

But do you notice something odd about this picture? If CC is a clone of Rainbow—an exact genetic copy—
then why are they different colors?

The answer lies in the X chromosome. In cats, a gene that helps determine coat color resides on this
chromosome. Both CC and Rainbow, being females, have two X chromosomes. (Males have one X and
one Y chromosome.) Since the two cats have the exact same X chromosomes, they have the same two coat
color genes, one specifying black and the other specifying orange.

Very early in her development, each of Rainbow's cells "turned off" one entire X chromosome, thereby
turning off either the black or the orange color gene. This process, called X-inactivation, happens normally
in females, in order to prevent them from having twice as much X-chromosome activity as males. It also
happens randomly, meaning that different cells turn off different X chromosomes.

So like all female mammals, Rainbow developed as a mosaic. Each cell that underwent X-inactivation gave
rise to a patch of cells that had one or the other coat color gene inactivated. Some patches specified black
other patches specified orange, and still others specified white, due to more complex genetic events. This is
how all calico cats, like Rainbow, get their markings.

CC looks different because she was made from a somatic cell from Rainbow in which the X-chromosome
with the orange gene had been inactivated; only the black gene was active. What's interesting is that, as CC
developed, her cells did not change the inactivation pattern. Therefore, unlike Rainbow, CC developed
without any cells that specified orange coat color. The result is CC's black and white tiger-tabby coat.

Left: CC (or Carbon Copy). Right: Rainbow. Photo courtesy TAMU, College of Veterinary Medicine.

Rainbow and CC are living proof that a clone will not look exactly like the donor of its genetic material.

Cloning livestock
Programs are underway to clone agricultural animals, such as cattle and pigs, that are efficient producers of
high-quality milk or meat.

A group of researchers at Utah State University led by Dr. Ken White, Dean of College of Agriculture &
Applied Science, have been able to clone steer from slaughterhouse carcasses. Their aim isn't to produce
animals for consumption—cloning is far more labor-intensive and expensive than conventional breeding
methods. Instead, they want to use these animals as breeding stock.

The important thing to know about beef cattle is that the quality and yield of their meat can be assessed
only after they are slaughtered. And male animals are routinely neutered when they're a few days old. That
is, their testes are removed, so they are unable to make sperm. But cells from a high-quality carcass can be
cloned, giving rise to an animal that is able, though conventional breeding methods, to pass its superior
genes to its offspring.

Scientists have also cloned mules, a reproductively sterile hybrid of a male donkey and a female horse;
dairy cows; and horses. One gelded racing horse, a male whose testes have been removed, has a clone that
is available for breeding. Some of the cloned cows produce about twice as much milk as the average
producer. And a cloned racing mule is ranked among the best in the world.
Drug production
Farm animals such as cows, sheep, and goats are being genetically engineered to produce drugs or proteins
that are useful in medicine. As an example, scientists could take cells from a cow that produces large
amounts of milk and grow them in culture. Then they could insert a gene into the DNA of these cells that
codes for a drug or a vaccine. If they take the nucleus from one of these cells and transfer it to a cow egg, it
could develop into a cow that makes the drug in its milk. Since every cell in the cow would carry the drug
gene, it could pass the gene to its offspring, creating a whole herd of drug-producing cows. Even better, we
could avoid the issue of the genetic reshuffling that happens during sexual reproduction and simply clone
our drug-producing cow.
Cloning Humans
The prospect of cloning humans is highly controversial, and it raises a number of ethical, legal, and social
challenges that need to be considered.

The vast majority of scientists and lawmakers view human reproductive cloning—cloning for the purpose
of making a human baby—immoral. Supporters see it as a possible solution to infertility problems. Some
even imagine making clones of geniuses, whose work could advance society. Far-fetched views describe
farms filled with clones whose organs are harvested for transplantation—a truly horrific idea.

For now, risks and technical challenges—as well as laws that make it illegal—will probably keep human
reproductive cloning from becoming a reality. Even though many species have been cloned successfully,
the process is still technically difficult and inefficient. The success rate in cloning is quite low: most
embryos fail to develop, and many pregnancies end in miscarriage.

Current efforts at human cloning are focused on creating embryonic stem cells for research and medicine,
as described above. However, many feel that this type of therapeutic cloning comes dangerously close to
human reproductive cloning. And once techniques become more streamlined and efficient, they fear that
some may be tempted to take that next step.

From a technical and moral standpoint, before human cloning becomes routine, we need to have a good
idea of the risks involved.