Published online 19 November 2008 |
Nature
456,
310-314
(2008)
| doi:10.1038/456310a
News Feature
Darwin 200: Let's make a mammoth
Evolution
assumes that extinction is forever. Maybe not. Henry Nicholls asks what
it would take to bring the woolly mammoth back from the dead.
Henry Nicholls
In
1990 the late Michael Crichton gave the idea of reviving extinct
species a slickly plausible and enormously entertaining workout in his
novel Jurassic Park. At that time the longest
genome that had ever been sequenced was that of a virus. The best part
of 20 years on, hundreds of animal genome sequences have been
published. This week, for the first time, the genome of something
undoubtedly charismatic and definitively extinct joins the list: the
mammoth (Mammuthus primigenius)1. If you want to bring a species back to life, the mammoth would be almost as dramatic as a dinosaur. And unlike Tyrannosaurus rex, the mammoth has close living relatives to lend a hand.
It
is a fair bet that a complete genome and closely related species would
make it easier to pull a Crichton on a mammoth than on a dinosaur. But
easier is far from easy. To put flesh on the bones of the draft
sequence to go from a genome to a living, breathing beast would
require you to master, at the very least, the following steps: defining
exactly the sequence or sequences you want for your creatures;
synthesizing a full set of chromosomes from these sequences; engulfing
them in a nuclear envelope; transferring that nucleus into an egg that
would support it; and getting that egg into a womb that would carry it
to term. None of those steps is currently possible. From getting a
definitive sequence to harvesting eggs from an elephant there are
all-but-insurmountable obstacles at every stage, and no evidence that
anyone is going to work very hard to solve them. But do any of them
actually make the dream impossible?
The sequence
The first stop in this mammoth challenge is to obtain a sequence good
enough for us to contemplate using it as the basis for a living being.
The sequencing of long-dead DNA such as that of mammoths uses fragments
at various levels of degradation. To detect and correct the base-pair
changes that can occur after death and to avoid the inevitable errors
involved in assembling millions of these tiny fragments into a coherent
sequence, it is necessary to sequence much more than a single genome's
worth of DNA. "If we want fewer than 1 error in 10,000 base pairs a
reasonable quality genome we would need to sample in the order of
12-fold coverage," says Svante Pääbo, director of the genetics
department at the Max Planck Institute for Evolutionary Anthropology in
Leipzig, Germany, who has worked on Neanderthal genomes2.
The genome published today has roughly 0.7-fold coverage. 'Reasonable
quality' for science does not mean the sort of genome you would want to
live with: in a human genome that error rate would mean 300,000
mutations.
Genome synthesis is further developed today than genome sequencing was when Crichton wrote Jurassic Park.
Coverage
can be improved as long as there's the money to do it, but old samples
offer particular challenges: a lot of contamination by bacterial,
fungal and other species' DNA. Thirty-five-fold coverage, which Pääbo
says is as good as it gets, would be "extremely costly and extremely
time-consuming", according to Eske Willerslev, head of the Ancient DNA
and Evolution Group at the University of Copenhagen. Ever-cheaper
sequencing, however, and the possibility of better preserved and
prepared samples, mean that those expenses of cost and time will
shrink. Willerslev sees nothing to stop researchers from producing a
mammoth genome as good as any genome today at some point in the future.
Whether such a genome would be good enough for a living being remains a
somewhat open question but with time and effort, it's plausible that
a sufficiently error-free genome can be arrived at.
A sequence
on its own, though, is not enough: researchers will need to work out
exactly how it divides up into chromosomes. The obvious solution would
be to tot up the number of chromosomes in an intact mammoth cell and
sift through the genomic data looking for their beginnings and endings.
But even the very best mammoth material falls short of this kind of
preservation (see 'You need to do more than thaw').
"We have no idea yet how many chromosomes mammoths had," says
Hendrik Poinar, a geneticist at McMaster University in Ontario, Canada.
Kerstin Lindblad-Toh, co-director of the genome sequencing and analysis
programme at the Broad Institute in Cambridge, Massachusetts, says that
the institute will release a sequence of the African elephant (Loxodonta africana) to seven-fold coverage some time in 2009. When they do, the mammoth geneticists will be all over it (see 'Let's fake a mammoth').
But it will take an immense amount of work to identify and locate all
the chromosome changes, gene deletions, duplications and rearrangements
that mammoths will have acquired since they diverged from their African
ancestors 7.6 million years ago. A sequence for the Indian elephant (Elephas maximus indicus), which is more closely related to the mammoth, would be of further help.
One
chromosome offers particular problems. In mammals the Y chromosome is
typically very small and hard to sort out, in part because it is
remarkably repetitive. Researchers have sidestepped the issue in the
elephant genome by sequencing a female. "The X chromosome is hard
enough to assemble and the Y chromosome is the hardest chromosome out
there," says Lindblad-Toh. The dinosaurs in Jurassic Park
were designed to all be female, to avoid unwanted breeding; cloned
mammoths might all be females, too, at least for the first generation,
just because it would be easier.
There are other repetitive
regions that will be hard to sequence with confidence, most notably the
centromeres, which help chromosomes to get where they are meant to go
in cell division. It is almost impossible to work out the centromeres'
exact sequence, says Bill Earnshaw of the Wellcome Trust Centre for
Cell Biology at the University of Edinburgh, UK. "You just get
hopelessly lost," he says. "It's like being in a forest where all the
trees look identical."
ILLUSTRATIONS BY: S. KAMBAYASHI
But
this need not be a sticking point, as artifice can make do for
accuracy. Just this year Earnshaw and his colleagues created a human
artificial chromosome that contained a synthetic but fully functional
centromere3.
In principle, that could work for a synthetic mammoth chromosome, says
Earnshaw. Replacements for the shorter repetitive sequences at the end
of chromosomes, called telomeres, are also doable. And although the
sequences will need specific sites at which chromosome replication can
start, too, Earnshaw says that "any long enough strand of DNA will have
sequences that can function as origins of replication".
Finally,
there is the question of genetic variation. Most published mammalian
genomes the mammoth draft included provide only a single version of
all the genes and other sequences in the genome. But mammals have two
versions of each gene one from their mother and one from their
father. Building a mammoth with chromosome pairs in which the two
chromosomes were identical would be a recipe for trouble, as the
effects of any bad gene would be felt to their fullest. Identifying
different versions of genes would add yet more to the sequencers' to-do
list, but it would be crucial to success.
DNA synthesis
With the genome sequenced in painstaking detail, glitches corrected,
chromosomes identified, key repeat sequences written in appropriately
and genetic variation introduced, it's time to turn towards the DNA
synthesis itself. The largest totally synthetic genome produced so far
is that of the bacterium Mycoplasma genitalium4.
This contained 582,970 base pairs; the mammoth weighs in at 4.7
billion, half as many again as are found in the human genome. Assuming
mammoths turn out to have the same number of chromosomes as elephants,
the task would be broken down into making 56 separate chromosomes, each
an average of some 160 million base pairs long.
Short
strings of bases that are made in the test tube can be assembled into
respectable double-stranded stretches of DNA about 8,000 base pairs
long without too much error, says Ralph Baric, a microbiologist at the
Carolina Vaccine Institute in Chapel Hill, North Carolina; a range of
companies will synthesise such sequences for less than a dollar a base
pair, and the reagents cost much less. But as neighbouring stretches
are joined together in vitro, the DNA molecules become increasingly unstable. The team that put together the M. genitalium
genome at the J. Craig Venter Institute in Rockville, Maryland, dealt
with this by inserting the unstable chunks of DNA into 'bacterial
artificial chromosomes'. The various components could then be stitched
together in the relatively welcoming environment of Escherichia coli. For the last steps, the researchers took the largest pieces assembled in E. coli
and inserted them into yeast artificial chromosomes, which are larger.
These were then recombined in living yeast cells to produce constructs
that had the entire genome in them.
This approach is
impressive in terms of speed and cost, says Drew Endy of the Department
of Bioengineering at Stanford University in California. But it's
unlikely to be scaled up to accommodate mammoth-sized chromosomes in
any straightforward way. After all, he says, at just over 12 million
base pairs, the entire yeast genome is much smaller than a medium-sized
elephant chromosome: "I would wonder if yeast could handle so much
exogenous DNA." This concern is echoed by Baric: "The bigger it is, the
quicker there are going to be nicks or breaks, allowing for degradation
or deletions in essential genes."
Even if it became possible
to synthesize a stable mammoth chromosome in sufficient quantities,
this would then have to be repeated for all the other chromosomes. "I
don't think we're going to be seeing any mammoths any time soon,"
concludes Baric. But it will not be long before synthetic biologists
develop a certain confidence in synthesizing microbial genomes from
scratch, he says. The technologies that are used to do so will, he
predicts, give a good indication of whether it might one day be
possible to reconstruct the genome of a large mammal such as a mammoth.
It is worth remembering that genome synthesis is further developed
today, in terms of the maximum lengths achieved, than genome sequencing
was when Crichton wrote Jurassic Park. And look how sequencing has progressed since then.
Wrapping it up
Once the chromosomes have been synthesized, they need to be put into a
nucleus. Cells wrap chromosomes back up into nuclei whenever they
divide, so this part of the process has been fairly well studied. In
the 1980s, researchers discovered that naked DNA added to extracts from
the eggs of frogs quickly becomes wrapped up in proteins that condense
it into chromatin; then membrane fragments bind and fuse to form a
functional nuclear envelope around the chromatin. "Such artificial
nuclei are capable of DNA replication and some DNA transcription," says
Douglass Forbes, professor of cell and developmental biology at the
University of California, San Diego. Forbes thinks that anyone trying
to make a mammoth nucleus in the foreseeable future would be best
advised to stick with frog extracts, rather than use fragments of the
nucleus from some more similar creature such as an elephant. "Mammalian
eggs, which are fertilized internally, might have much less ability to
assemble nuclei," she says. When later transferred into the cytoplasm
of an elephant cell, elephant nuclear proteins might complement or
replace the frog-specific counterparts used to make the mammoth
pseudo-nucleus, she says, giving it a more properly mammalian look and
feel.
It
would also be a challenge to ensure that the nuclear membrane engulfs
all the mammoth chromosomes. "You will somehow need to keep them
together when you inject them," says Eric Schirmer at the Wellcome
Trust Centre. If you don't, miniature nuclei may form that contain a
random rabble of chromosomes. The way these chromosomes sit with
respect to one another might also affect gene expression; how to
engineer the correct configuration, nobody knows.
Egg collection
It's almost time to contemplate the vagaries of nuclear transfer, but
not before you have sourced your elephant eggs, and these are likely to
be in pretty short supply. Female elephants ovulate on a 16-week cycle,
although they regularly skip five or so years owing to gestation and
lactation. Stopping these natural breaks in cycling would be both cruel
and unproductive; cow elephants that don't gestate have a strong
tendency to develop massive ovarian tumours. When they do ovulate, only
one oocyte is released; a litter of little elephants would be a death
sentence, and even twins are remarkably rare.
On the positive
side, though, elephants' infrequent ovulations are preceded by an early
warning; uniquely among mammals that have been studied to date,
elephant ovulation involves not one but two surges in luteinizing
hormone, separated by 1820 days. "The first hormone peak dissolves the
vaginal mucous and forwards just one follicle for development," says
Thomas Hildebrandt of the Institute for Zoo and Wildlife Research in
Berlin, Germany. "The second peak stimulates ovulation."
In
other creatures it would be quite straightforward to get the follicle
in which an egg is developing out of the ovary after this surge of
harbinger hormones; you use ultrasound to guide a harvesting implement
up the reproductive tract, or perform a laparoscopy, during which the
abdominal cavity is inflated to make room for the job to be done
surgically.
Unfortunately,
quirks of elephant biology rule out both these approaches. Whereas the
entrance of the vagina is pretty simple to locate in most mammals,
elephants have more than a metre of urogenital canal between the
outside world and the hymen. This canal is as far as a bull-elephant's
penis gets; the hymen remains intact even after copulation, which may
be an evolutionary hangover from the elephant's aquatic past.
Hildebrandt and his colleagues have developed a way to navigate an
instrument up the canal, through the tiny aperture in the hymen that
lets sperm in, along the vagina and into the uterus; they use it to
perform artificial insemination with sex-selected sperm. But
Hildebrandt says that even with such instruments threaded into the womb
it would be almost impossible to locate a single mature follicle
without some extra guidance, and the ovaries are too deep inside the
abdominal cavity for the precise position of the follicle to be
revealed by ultrasound. Laparoscopic surgery is also out of the
question, as elephants have no pleural space between their body wall
and lungs. "Inflating the abdominal cavity during laparoscopy would
compress the lungs and kill the animal," says Hildebrandt.
There
may, however, be an ingenious way out of this bind. Cryobiologists have
in the past transplanted tissue from the ovaries of one animal into the
ovaries of another. The procedure has worked even if the tissue had
been frozen and thawed out between leaving the donor and being grafted
into the recipient. Not only has this work led to the treatment of
infertility after chemotherapy, it has also raised the possibility that
ovarian tissue from endangered species could be transplanted into
laboratory animals, making them a source of eggs-on-demand. For
Hildebrandt, the only realistic way of getting a steady source of
elephant eggs would be to collect tissue from the ovary of a recently
deceased elephant and graft slivers of it into a laboratory mouse or
rat with a suppressed immune system that won't reject them.
The urogenital canal is as far as a bull elephant's penis gets.
This
has in fact already been done. In the 1990s, frozen samples of ovarian
tissue from African elephants culled in South Africa's Kruger National
Park were thawed and transplanted into mice, and afterwards researchers
saw what seemed to be mature follicles develop5.
"But we didn't have enough material to assess whether those eggs were
competent," recalls John Critser, now professor of comparative medicine
at the University of Missouri in Columbia, who led the study.
Although
the elephant work has gone no further, researchers have managed to
produce live and apparently healthy offspring from eggs that have come
from tissue transplanted into another species. Such successes, though,
have been with closely related species rats acting as hosts for mouse
tissue6,
for example. It's harder to see how elephant eggs would mature
successfully in a mouse, not least because the mouse's oestrus cycle
lasts just 46 days. It would probably be necessary to remove the
mouse's pituitary gland and subject it to a hormonal cycle that
approximates an elephant's with hormone treatments, says Critser. "It's
not something that you could use easily right now for the production of
oocytes. But that's not to say that it couldn't be developed."
Nuclear transfer
With
a ready supply of eggs, it should not take long to perfect techniques
for removing the native elephant nucleus to make space for the
synthetic mammoth nucleus. But that might not be all the preparation
needed for the nuclear transfer. The mitochondria organelles that
provide cells with energy through respiration have their own genomes,
specific to each species. Hybrids with a nucleus from one species and
mitochondria from another can be viable an embryo with sheep
mitochondria but nuclear DNA from a mouflon developed into an animal,
called Ombretta, in 2001 (ref. 7)
but there are risks of incompatibility. The close evolutionary
relationship between elephants and mammoths reduces the chances of
incompatibility, but Stefan Hiendleder, professor of animal science at
the University of Adelaide in Australia, cautions that there would
still be risks: human cells that have had their mitochondria replaced
with those from other primates have failed to show proper respiration8.
This means that researchers might need to rustle up some synthetic
mammoth mitochondria and insert them into the elephant cytoplasm. With
the mammoth mitochondrial genome already well sequenced9,
this should be relatively simple. And all the elephant mitochondria
would have to be cleaned out, in case they hybridize with the
newcomers, warns Hiendleder.
Nuclear transfer remains a fickle
and inefficient way of producing new mammals even without such
concerns. Only a few of the transfers result in embryos and not all of
those embryos manage to establish a placenta. Of those that do, many
abort spontaneously and the few successful live births frequently have
developmental abnormalities.
But it is reasonable to hope that
things may get better, says Xiuchun Tian of the Center for Regenerative
Biology and Department of Animal Science at the University of
Connecticut in Storrs. The most likely explanation for the inefficiency
of nuclear transfer is that the genes are not being expressed in a
manner appropriate for embryonic development. This, says Tian, is
probably down to errant 'epigenetic' signals inherited patterns of
DNA methylation, histone modification, microRNA presence and chromatin
structure, all of which can have a crucial effect on gene expression10.
The recent discovery that the nuclei of normal body cells can be
induced into an embryonic-like state without the need for nuclear
transfer will help researchers to understand the effect of these
epigenetic signals and how to manipulate them, she says.
Better
still, there may be an alternative way to dress up a synthetic nucleus
in suitably epigenetic garb. Even if an early embryo is doomed not to
go all the way to term, it can still be used as a source of stem cells.
These could then be introduced into normal elephant embryos to create
chimaeras in which some cells are mammoth and some elephant. Such
chimaeras may stand a better chance of developing to term, and although
they wouldn't be mammoths, they would be a way by which mammoths might
then be made.
If enough chimaeras are created in this way,
some should end up with mammoth cells in their ovaries or testes,
giving you elephants that produce mammoth eggs or sperm. 'Germ-line
chimaeras' of this type have already been created in several species.
In 2004, Japanese scientists11
made salmon with trout cells in the testes that produced sperm capable
of fertilizing trout eggs and producing bona fide baby trout.
Gametes
from germline chimaeras would be much more likely to be properly
imprinted, says Tian indeed, once you have a supply of mammoth eggs
and mammoth sperm you might well consider the project close to bearing
fruit.
Embryo transfer
With a fertilized mammoth egg either made through direct nuclear transfer or through the in vitro
fertilization of mammoth eggs with mammoth sperm from chimaeras the
mammoth challenge comes to its final stage. Although the
inaccessibility of elephant eggs means that nobody has ever performed
elephant embryo transfer, Hildebrandt has thought about what it would
take. First, he recommends inserting an arm up the urogenital canal to
inject some sperm-free elephant ejaculate in the direction of the hymen
on the day that hormones reveal the elephant is ovulating. "We think
the sperm-free component is needed to prepare the female's uterus to
receive an embryo," he says. After that, the transfer of the cloned
mammoth embryo into the uterus, a total distance of some 2.5 metres,
should be possible using the same apparatus used for artificial
insemination, says Hildebrandt. "We are coming quite close to the
oviduct, which would be the place to put a cloned embryo back into an
elephant." An embryo at the four-cell stage would need to be
transferred about two days about ovulation, he says.
At that point, the last concern becomes whether a mammoth fetus
would be suited to the uterus of its surrogate mother. Evidence from
preserved mammoths suggests that size, at least, should not be a
problem. An Indian elephant calf can weigh around 120 kilograms at term
and stands around 1 metre tall. Dima a famous mammoth calf unearthed
in northeastern Siberia in 1977 is estimated to have been about the
same mass and height when he died aged 78 months. It's a similar story
for Lyuba, a calf discovered in Russia's Yamal Peninsula last year.
Although Lyuba's mass when she died has not yet been definitively
ascertained, initial reports suggest she was only 90 centimetres tall
and 4 months old, says Daniel Fisher, professor of ecology and
evolutionary biology at the University of Michigan in Ann Arbor and one
of an international team poring over her spectacularly preserved
remains. Most evidence indicates that newborn woolly mammoths were
about the same size as newborn elephants, he says. "They could even
have been smaller." Mercifully, we probably need not concern ourselves
with how to incubate a preterm mammoth fetus.
Birth and after
A
single artificial mammoth would be a freak, not a species; once she was
born others including, ideally, males would have to follow. Their
genomes would have to be tweaked to ensure a certain diversity. A place
for them to live would have to be found, as would an ecosystem into
which they could integrate. It would be a huge undertaking just as
synthesizing mammoth chromosomes and reprogramming them into
embryo-friendly nuclei would be. Perhaps the whole idea will remain too
strange, too expensive, too impractical, even too unappealing for
anyone to take seriously. But the fact that just 15 years ago cloning
mammals was confidently ruled out by many as being impractical should
give people pause before saying any such thing is impossible. On
Darwin's 200th birthday in 2009, reoriginating extinct animal species
will still be a fantasy. By 2059, who knows what may have returned,
rebooted, to walk the Earth?
Henry Nicholls is a science writer who lives in Greenwich, England. His most recent book is Lonesome George.
http://www.nature.com/news/2008/081119/full/456310a.html