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Will Cloning Ever Save Endangered

Animals?
Right now, cloning is not a viable conservation strategy. But some researchers remain
optimistic that it will help threatened species in the future
By Ferris Jabr | March 11, 2013

James and snowmanradio, Wikimedia Commons

In 2009 the Brazilian Agricultural Research Corp. (Embrapa) and the


Brasilia Zoological Garden began scavenging and freezing blood, sperm
and umbilical cord cells from roadkill and other wild animals that had
died, mostly in the Cerrado savannaanincredibly diverse collection of
tropical forest and grassland ecosystems home to at least 10,000 plant
species and more than 800 species of birds and mammals, some of which
live nowhere else in the world. Specimens were collected from the bush
dog, collared anteater, bison and gray brocket deer, among other species.
The idea was to preserve the genetic information of Brazil's endangered
wildlife. One day, the organizations reasoned, they might be able to use
the collected DNA to clone endangered animals and bolster dwindling
populations. So far the two institutions have collected at least 420 tissue
samples. Now they are collaborating on a related project that will use the
DNA in these specimens to improve breeding and cloning techniques.
Current cloning techniques have an average success rate of less than 5

percent, even when working with familiar species; cloning wild animals is
usually less than 1 percent successful.
Any animals born during Brazil's new undertaking will live in the Brasilia
Zoo, says Embrapa researcher Carlos Martins. Expanding captive
populations of wild animals, he and his team hope, will discourage zoos
and researchers from taking even more wild animals out of their native
habitats. Martins and his colleagues have not yet decided which species
they will attempt to clone but the maned wolf and jaguar are strong
candidates. The International Union for Conservation of Nature classifies
both animals as "near threatened" on its Red List of Threatened Species,
two levels below "endangered."
Many researchers agree that, at present, cloning is not a feasible or
effective conservation strategy. First of all, some conservationists point
out, cloning does not address the reasons that many animals become
endangered in the first placenamely, hunting and habitat destruction.
Even if cloning could theoretically help in truly desperate situations,
current cloning techniques are simply too ineffective to make much of a
difference. Compared with cloning domestic speciesparticularly cattle,
which have been successfully cloned for years to duplicate desirable traits
cloning endangered species is far more difficult for a number of reasons.
Successful cloning generally involves at least three essential components:
DNA from the animal to be cloned; a viable egg to receive that DNA; and a
mother to gestate the resulting embryo. Often, hundreds of embryos and
attempted pregnancies are needed to produce even a few clones. Scientists
usually have a poor understanding of endangered animals' reproductive
physiology, which makes it too risky to extract a sufficient number of eggs
from that species or rely on females of that species to give birth to clones.
Legal protections sometimes preclude threatened species from such
procedures as well. To compensate, researchers fuse the DNA of an
endangered species with eggs from a closely related species and select
mothers from the latter. Such hybrid embryos often fail to develop
properly.

Although they are keenly aware of these problems, Martins and his
colleagues, as well as a few other scientists around the world, think that
efforts to archive the genetic information of endangered wildlife are
worthwhile. Some researchers remain optimistic that cloning will become
a useful tool for conservation in the future. Optimists point to recent
successes cloning wild mammals using closely related domestic species,
improved techniques for preventing developmental abnormalities in a
cloned embryo, better neonatal care for newborn clones and in vitro
fertilization made possible by stem cells derived from frozen tissue.
The first clones
In the early 1950s, at the Lankenau Hospital Research Institute in
Philadelphia, Robert Briggs and Thomas King successfully cloned 27
northern leopard frogs through a process known as nuclear transfer. The
nucleus, often called the command center of the cell, contains most of a
vertebrate's DNAexcept for the DNA within bean-shaped, energygenerating organelles named mitochondria. Briggs and King emptied frog
eggs of their nuclei, sucked nuclei out of cells in frog embryos and injected
those nuclei into the empty eggs. Many of the eggs developed into
tadpoles that were genetically identical to the embryos that had donated
their nuclear DNA.
In 1958 John Gurdon, then at the University of Oxford, and
colleagues cloned frogswith nuclear DNA extracted from the cells of fully
formed tadpoles. Unlike embryonic cells, which are genetically flexible
enough to become a variety of different tissues, a tadpole's cells are
"differentiated"that is, the patterns of genes they express have changed
to fit the profile of a specific cell type: a skin, eye or heart cell, for
example. Gurdon demonstrated that, when transplanted into an egg,
nuclear DNA from a mature cell reverts to the more versatile state
characteristic of DNA in an embryo's cells. This breakthrough encouraged
scientists to try cloning far larger animals using DNA from adult cells.
In 1996 researchers in Scotland attempted to clone a female Finn-Dorset
sheep. They injected nuclei extracted from her udder cells into nearly 300
empty eggs derived from Scottish blackfaces, a different sheep breed. Out

of those prepared eggs, the scientists managed to create more than 30


embryos. Only five of those embryos developed into lambs after being
implanted in surrogate Scottish blackfaces. And only one of those lambs
survived into adulthood. The researchers named her Dolly.
Since then some biologists have repeatedly suggested that cloning could
help save endangered species, especially in dire situations in which only a
few dozen or a handful of animals remain. The smaller, more homogenous
and more inbred a population, the more susceptible it is to a single
harmful genetic mutation or disease. Clones could theoretically increase
the genetic diversity of an endangered population if researchers have
access to preserved DNA from many different individuals. At the very
least, clones could stabilize a shrinking population. And, some researchers
argue, a genetically homogenous but stable population would be better
than extinction; some highly inbred groups of wild animals, such
as Chillingham cattle in England, have survived just fine for hundreds of
years.
One species that might benefit from cloning is the northern white
rhinoceros, which is native to Africa. In 1960 the global northern white
rhino population was more than 2,000 strong, but poaching has reduced
their numbers to as few as 11 today. By last count, three live in zoostwo
in San Diego and one in the Czech Republicfour live in the Ol Pejeta
Conservancy in Kenya and as few as four individuals may still live in the
wild based on unconfirmed reports, but they have not been spotted in
several years. Most of the captive animals are uninterested in mating or
infertile, although two rhinos mated in the summer of 2012.
Right now, though, cloning is unlikely to help the white rhino or any other
threatened species. To date, the story of cloning endangered animals is
one of a few high-profile successes and many, many failures. Since the
early 2000s, using the same technique that produced Dolly, researchers
have cloned several endangered and even extinct mammals, including
a mouflon sheep and a bovine known as a gaur in 2001; a kind of wild
cattle called a banteng in 2003; a wild goat known as the Pyrenean ibex in
2009; and wild coyotes in 2012. In each case many more clones died

before birth than survived; in most cases none of the clones survived into
adulthood.
Mismatched
All those attempted clones of endangered or extinct animals died in
different ways for different reasons, but they all shared one fundamental
problemthey were not exact replicas of their counterparts. In most
cases, researchers have combined DNA from the threatened species with
eggs from a related domestic species. Each surrogate mother is often
implanted with dozens of hybrid embryos in order to achieve at least a few
pregnancies, a strategy that requires extracting hundreds of eggs. Because
the reproductive physiology of most endangered animals is so poorly
understood, researchers are often unsure when the animals ovulate and
how best to acquire their eggs. In some cases legal protections prevent
scientists from harvesting eggs from threatened species. For all these
reasons, they turn to more familiar domestic species instead.
Injecting the DNA of one species into the egg of another specieseven a
closely related onecreates an unusual hybrid embryo that often fails to
develop properly in the womb of a surrogate mother. Hybrid embryos
have the nuclear DNA of the cloned species and the mitochondrial
(mtDNA) DNA of the donor egg. This mismatch becomes problematic as
the embryo develops. Nuclear DNA and mtDNA work together; they both
contain genetic recipes for proteins with which cells extract energy from
food. In a hybrid embryo these proteins do not always fit together
properly, which leaves cells starved for energy. Complicating matters
further, the surrogate mother often rejects the hybrid embryo because she
recognizes some of the embryo's tissues, particularly the placenta, as
foreign.
Another problemand the most intractable so faris that a hybrid
embryo created via nuclear transfer is not a genetic blank slate like most
embryos. All vertebrates begin life as hollow balls of embryonic stem cells,
which can become almost any type of adult cell. Each of those stem cells
contains a copy of the exact same genome packaged into chromosomes
tight bundles of DNA and histone proteins. As the embryo develops, the

stem cells begin to take on their adult forms: some become skin cells,
others heart cells and so on. Different types of cells begin to express
different patterns of genes. Inside each cell an assortment of molecules
and enzymes interacts with DNA and histones to change gene expression.
Some molecules, such as methyl groups, physically block cellular
machinery from reading the genetic instructions in certain segments of
DNA; some enzymes loosen the bonds between histones and DNA,
making particular genes more accessible. Eventually, each cell typeskin
cell, liver cell, brain cellhas the same genome, but a different
epigenome: a unique pattern of genes that are actively expressed or
effectively silenced. Over time, an adult cell's epigenome can change even
further, depending on the animal's life experiences.
So when researchers inject an adult cell's nucleus into an empty egg, the
nucleus brings its unique epigenome with it. As Gurdon's early
experiments in the 1950s and subsequent studies have shown, an egg is
capable of erasing the epigenome of introduced nuclear DNA, wiping the
slate cleanto some extent. This process of "nuclear reprogramming" is
poorly understood, and the egg often fails to complete it properly,
especially when the egg is from one species and the nuclear DNA from
another. Incomplete nuclear reprogramming is one of the main reasons,
scientists think, for the many developmental abnormalities that kill clones
before birth and for the medical issues common to many survivors, such
as extremely high birth weight and organ failure.
Some researchers see ways around these problems. Pasqualino Loi of the
University of Teramo in Italy was part of a team that successfully cloned
endangered mouflon sheep in the early 2000s; the clones died within six
months of birth. Loi and his colleagues think they can increase the
chances of a hybrid embryo surviving in a surrogate mother's womb. First,
they propose, researchers could nurture a hybrid embryo for a short time
in the lab until it develops into what is known as a blastocystthe ballshaped beginnings of a vertebrate composed of an outer circle of cells, the
trophoblast, surrounding a clump of rapidly dividing stem cells known as
the inner cell mass. Eventually, the trophoblast becomes the placenta.
Researchers could scoop out the inner cell mass from the hybrid

blastocyst, Loi suggests, and transplant it into an empty trophoblast


derived from the same species as the surrogate mother. Because the
surrogate mother is far less likely to reject a trophoblast from her own
species, the developing embryo within has a much better chance of
surviving.
Scientists have also figured out how to encourage nuclear reprogramming
by bathing the egg in certain compounds and chemicals, such as
trichostatin A, which stimulate or inhibit the enzymes that determine a
cell's epigenome. Most recently, Teruhiko Wakayama of the RIKEN
Center for Developmental Biology in Kobe, Japan and his colleagues
produced 581 cloned mice from a single donor mouse over 25 generations,
using trichostatin A to achieve success rates as high as 25 percent in some
but not all generations. To solve the mismatch of mtDNA and nuclear
DNA, Loi suggests simply removing the egg's native mtDNA and replacing
it with mtDNA from the species to be clonedsomething that researchers
tried in the 1970s and '80s, but have not attempted recently for reasons
that are unclear.
Some of the most successful attempts to clone endangered animals in
recent years have involved two of the most beloved domestic speciescats
and dogs. At the Audubon Center for Research of Endangered Species in
New Orleans, Martha Gomezand her colleagues have created
many African wildcat clones since the mid-2000s, using domestic cats as
surrogate mothers. Gomez says eight clones have survived into adulthood
so far and are all healthy today. She attributes her success, in part, to the
fact that wildcats and domestic cats are much more closely related to each
other than are most wild and domestic species paired for the purpose of
cloning. She and her team have also learned to increase success rates with
caesarian sectionsto spare clones the stress of a typical birthand to
keep newborn clones in intensive care for a few weeks, as though they
were premature babies. In 2008, B. C. Lee of Seoul National University in
Korea and his colleagues achieved similar success using domestic dogs to
create three healthy male gray wolf clones. Lee's team had previously
created two female gray wolf clones. All five animals survived into
adulthood, Lee confirms.

Working with black-footed cats, which are native to Africa and listed as
"Vulnerable" on the Red List, Gomez is now focusing on a method of
cloning that differs from nuclear transfer. She is trying to transform adult
cells from black-footed cats into stem cells and subsequently induce those
stem cells to become sperm and eggs. Then, through in vitro fertilization
or similar techniques, she could impregnate domestic cats with blackfooted cat embryos. Alternatively, stem cell-derived sperm and eggs could
be used to impregnate females of the endangered species.
To say that this approach is technically challenging would be an
understatement, but researchers have made impressive progress. In 2011
Jeanne Loring of the Scripps Research Institute in La Jolla, Calif., and her
colleagues produced stem cells from the frozen skin cells of two
endangered speciesthe northern white rhino and a baboonlike primate
known as a drill. And in 2012 Katsuhiko Hayashi of Kyoto University
Graduate School of Medicine and colleagues turned skin cells from adult
mice into stem cells, which they then transformed into viable eggs. After
fertilizing the eggs with sperm in test tubes, the researchers implanted the
embryos in surrogate mother mice that gave birth to healthy and fertile
offspring.
"I'm not saying cloning is going to save endangered species," Gomez says,
"but I am still a believer of cloning as another tool. It's not easy, though.
The research moves slow."
Teramos Loi remains optimistic too. He thinks that scientists should
continue to collect and preserve the genetic information of endangered
animals, as Brazil has done, creating bio-banks of tissue on ice, such as
the "frozen zoo" at the San Diego Zoos Institute for Conservation
Research. If researchers manage to dramatically increase the efficiency of
cloning wild and endangered animalswhether with nuclear transfer or
in vitro fertilizationthen the DNA they need will be waiting for them. If
they do not, bio-banks will still be useful for more basic research. "Once
cloning of endangered animals is properly established, it will be a very
powerful tool," Loi says. "If something can be done, it will be done in 10

years."