De-Extinction: Making Jurassic Park a Reality
Introduction
The process of bringing back extinct species, most commonly known as de-extinction, used be
nothing more than a science fiction dream, popularised in entertainment media such as Michael
Chrichton’s Jurassic Park (1990) series. However, during this revolution in biotechnology and genetic
manipulation techniques in recent years, species such as the mammoth and the American Passenger
Pidgeon being brought back to life could be quite the stark reality. De-extinction can be described as
a process in which an organism that has been extinct has been reborn, or alternatively a species
created that greatly resembles an extinct species is born (Martinelli, Oksanen et al. 2014). In the
public eye, this idea of bringing extinct species back to life is becoming increasingly more prevalent,
as high profile discoveries such as Yuka, the most well preserved mammoth ever found in glacial ice,
is drawing the media’s interest, along with the idea of cloning the animal from its incredibly well
preserved tissue http://www.bbc.co.uk/nature/17525070. Other types of animals that have high
interest in being brought back include the moa, the Carolina parakeet and the American Passenger
Pigeon, to name a few. Another example of its increasing popularity can be seen through popular
mainstream literature such as the National Geographic devoting a cover story to this area of science,
as well as establishments such as the Revive and Restore Network gaining support from both TED
and the National Geographic Society holding TED conferences further explaining the science and
possibilities behind it http://tedxdeextinction.org/.
As of yet, there is little to no papers published upon de-extinction as this is still a very new science.
However, the underlying experiments which is incorporated into the process of de-extinction is well
documented. These include processes such as Interspecies germ cell transplantation, which involves
using primordial germ cells (PGCs) from one species and genetically altering them to produce
gametes that create another species and putting it back into the host animal (van de Lavoir,
Diamond et al. 2006). This causes the germline of these chimeric animals to change to another
related species and allows them to give birth to species that lie out with their own. Another option is
to use new gene editing techniques such as the CRISPR-Cas9 system to specifically target genes of
related species of extinct animals and manually alter genes to express phenotypes that the extinct
species would have (Hsu, Lander et al. 2014). Both of these techniques will be explored in greater
detail later in this essay. Finally using classic cloning techniques using nuclear transfer of intact nuclei
of cells into an enucleated oocye could also be an option if an intact nuclei with intact DNA were in
existence for an extinct species (Solter 2000)
Although one can naturally get excited about the prospect of bringing back animals that were once
lost to the world, this brand new avenue of science contains a number of practical problems of using
cloning techniques, as well as a variety of ethical issues such as ecological benefit, bio-objectification
concerns and indeed whether it is right to bring back extinct species at all if they were unfit to
survive? In this essay, a critical review shall be used on both the science and the variety of issues
that could inhibit the development of this science into a true reality in the very near future.
Germline Manipulation
Primordial germ cells (PGCs) can be described as the initial precursors to the production of
sperm and eggs (van de Lavoir, Diamond et al. 2006). In most animals, the migration of the
germ line from their somatic origins is one of the earliest events that occur during
development (Blackler, Gecking 1972). The fact that PGCs tend to occur separately from
their gonadal end goal for migration and specification, this allows the removal of these PGCs
and allow the genetic editing of the original PGCs. Also it allows the reintroduction of PGCs
from another species, which would then cause chimeric animals that would produce
genetically altered offspring or indeed, entirely new species through reproduction with
another altered animal of the opposite sex. This process can be most easily carried out in
avian embryos, as PGCs can be first identified in an extra-embryonic region known as the
germinal crescent in the epiblast at around 18 hours of incubation. Then at around 50-55
hours of development, PGCs migrate and reach the developing gonad through the
circulatory system, allowing the production of functioning sperm and oocytes (van de Lavoir,
Diamond et al. 2006, Kuwana 1993). By removing the PGCs from one embryo and replacing
them with PGCs from another, this would create a chimeric strain of that species with the
ability to produce offspring from the second species.
An experiment conducted by (Liu, Khazanehdari et al. 2012) aimed to create transgenic
chimeric ducks (Anas domesticus) with chicken (Gallus gallus domesticus) PGCs that would
go on to produce chicken offspring. There are several distinct phylogenic differences
between the chicken and the duck. Ducks as waterfowl, belong to the order of
Anseriformes, whilst chickens belong to the order of Gallinformes. In order for these
chimeric ducks to produce chick offspring, the injected PGCs would be required to migrate
to the gonads of the duck embryo, proliferate and then successfully differentiate into
functional gametes in the gonads of the duck. It was shown that chicken DNA had reached
the gonad of the developing duck embryo a week before hatching by using specific chicken
and WT probes via in-situ hybridisation. However, upon assessing the duck semen, it was
seen that only 20.2% of total sperm produced by the ducks were of chicken origin, which
naturally inhibits the fertility of the chimeric duck. Female chickens were then artificially
inseminated with the chimeric duck semen, which produced 2 pure chicken embryos out of
367 eggs, giving a fertility rate of
0.8% and reached full maturity
after 4 months (see Figure 1).
Albeit the experiment was a
success in terms of producing an
interspecies offspring, the
frequency at which viable
embryos were born was
incredibly low. This could be due
to the distance in evolution
between the duck and the
chicken, being from entirely
Fig 1. The chicken offspring that was produced by chimeric male ducks (Liu,
Khazanehdari et al. 2012). (a)The male chimeric duck (Wd25), female
chicken (BR131) and their chicken offspring (wdp001). (b) Three chicken
offspring (wdp003, wdp002, wdp004) from male chimeric ducks. (c) The
progeny of a chimeric duck derived chicken (wdp001) after insemination
with rooster semen. (d) Molecular sexing test of PGC offspring. (e) Species
identification of the offspring with chicken-specific and duck specific
primers. (f) PCR sensitivity test on detecting chicken sperm from a mixture
of chicken and duck semen. (g) Detection of chicken sperm from chimeric
duck semen with chicken-specific and duck specific primers
different orders in the evolutionary timeline. As a result, exogenous germ cells can prove
incompatible with Sertoli cells due to the differences in micro environmental interactions
between the duck and the chicken in this case, severely inhibiting the frequency of
successful fertilisation. In a high output animal such as avian birds for eggs, a positive
outcome of this type of experiment clearly occurs fairly swiftly. However if the same
methods were used in animals that produce less offspring such as large mammals, it could
prove far more difficult at such a low fertility rate as shown here. Naturally, the closer
related a species is to the other inevitably improves its chances, but there is still the
problem of the incompatibility between any two species when you integrate different cell
lines of any sort. Another problem with using non avian PGCs is the ease of the extraction of
PGCs. For example, mouse PGCs can be found at the posterior end of the primitive streak
and in the allantois, a far more difficult area for PGC extraction compared to the epiblast, as
well as the obvious problem of then putting the chimeric embryo back into the mother’s
uterus for in utero development to occur. However, when successful, this method of germ
line manipulation does have the immediate advantage of propagating endangered species.
For example, the endangered Houbara Bustard was successfully produced in chimeric
chicken producing bustard sperm (Wernery, Liu et al. 2010). Using similar analysis
techniques, it was shown that out of 45 Houbara eggs, one surviving male live born Houbara
bustard, shown to be of pure line. Although it encountered similar difficulties as the
chicken-duck experiment in terms of fertility, the fact that an endangered species of bird
was being produced in chickens – an animal that can produce fertile eggs non-seasonally
throughout the entire year is of considerable advantage to the preservation of the Houbara
buzzard species.
In terms of its effectiveness in its use to bring extinct species back from the dead, this
method would prove useful in bringing back avian species such as the American passenger
pigeon. By removing the PGCs from the most related species of pigeon to the passenger
pigeon, and then removing the DNA or nucleus from the PGC and replacing it with intact
DNA or nuclei of the passenger and reintroducing it to the host animal could in theory
produce a chimera capable of creating pure line passenger pigeons. However the
survivability of the altered PGCs could also be affected by the introduction of exogenous
DNA into the cells, which could cause further migratory and proliferative problems on top of
the initial downfalls of this type of experiment that has been covered previously. This type
of alteration might be better suited to genetically editing the PGCs to maintain their initially
endogenous status, presuming that the alterations made to the PGCs to turn them into
something similar to the species in question that was to be resurrected doesn’t affect its
ability to function as a PGC.
CRISPR-Cas-9 Genomic Editing
Instead of using a de-extinction method that requires the insertion of an existing genome, it
could be possible to alter the DNA of highly related surviving species and editing it to create
an animal that is genetically identical to an extinct species. Now with the introduction of the
CRISPR-Cas-9 (clustered regularly interspaced short palindromic repeats) genomic editing
technology, we can accurately delete, insert and manipulate genes endogenously in any
organism one chooses (Hsu, Lander et al. 2014). Using this targeted genome editing
technique, we can use customisable Cas9 nucleases allowing for the targeted deletions,
insertions and sequence changes in nearly all organisms and cell types. For any form of
targeted genome editing, the creation of a double stranded DNA break (DSB) is vital for the
genomic locus to be modified (Carroll 2011). When this occurs using techniques such as
CRISPR, the break can be repaired via two methods: non-homologous end-joining (NHEJ)
and homology directed repair (HDR). Using NHEJ, whereby the break in the DNA leaves
exposed ends of DNA on the broken strands can be used to add insertional/deletion
mutations of varying lengths, thus disrupting the reading frame for a coding sequence or
binding sites for trans-acting factors in genetic sequences such a promoters and enhancers
(Sander, Joung 2014). HDR repair uses specific point mutations in desired sequences
through the recombination of target sites using exogenous DNA templates. Before CRISPR
became the mainstay in genomic editing in 2013, zinc fingers (ZFN) and transcription
activator like effector nucleases (TALENs) but each had a variety of advantages and
disadvantages.
CRISPR’s natural role in bacteria
and archaea was as an adaptive
immune system to defend itself
from foreign nucleic acid such as
viruses (Mojica, Diez-Villasenor et
al. 2000). Type II CRISPR systems
use sequences from invading DNA
between CRISPR repeat
sequences which are encoded as
arrays within bacteria (Fig. 2a).
These CRISPR repeat arrays are
then broken down further into
smaller CRISPR RNA (crRNA).
These small segments of RNA act
as a variable sequence which is
transcribed from invading DNA
called a protospacer
sequence as well as the rest
Figure 2. (a) Naturally occurring CRISPR system as an immunoresponse technique
of the CRISPR repeat. Each
whereby foreign DNA sequences become CRISPR arrays, creating crRNAs bearing
protospacer regions that are complementary to invading DNA. These then hybridise to
crRNA hybridises with a
tracrRNA which then complex with Cas9 nuclease which then cleave the invading DNA
at the areas in which the protospacer was translated. (b) An example of engineered
CRISPR-Cas system using a combination of crRNA and tracrRNA sequence. The single
gRNA complexes with Cas9 to control cleavage at targeted DNA sites. (c) An example of
a crRNA/tracrRNA complex and gRNA. Adapted from (Sander, Joung 2014)
second section of RNA, known as the transactivating CRIPR RNA (tracrRNA). These two
sections of RNA complex with Cas9 to then cleave target DNA sequences from 3 base pairs
from short non coding sequence known as protospacer adjacent motifs (PAM).
An example of a genetic engineered model of this system incorporates the type II CRISPR
system from S. pyrogenes for creating DB and genome editing. In the simplest form of the
system, the Cas 9 nuclease and a guide RNA (gRNA) – a fusion of crRNA and tracrRNA – is
incorporated into cells of a target organism such as an ECS for manipulation purposes (Fig
2b). Twenty nucleotide at the 5’ end of the gRNA act as the protospacer to direct Ca9 to
specific target DNA site using classic base pairing rules. These sequences lie immediately 5’
of the PAM sequence that matches the form 5’-NGG. Therefore Cas9 will be directed to all
areas of DNA of the form N20-NGG by altering the first 20nt of the gRNA to align with
specific target sequences.
The result of creating DBSs is that in the repair of these breaks causes high fidelity in the
original structure, causing random and generally undesirable mutations, lest the technique
is being used for specific gene knockout. However, using Homology Directed Repair (HDR)
techniques allows specific genetic mutations of choice (Wang, Yang et al. 2013). This process
involves electoporating or injecting cells with Cas9, sub genomic RNA (sgRNA) and a single
stranded DNA oligonucleotide which is homologous to the breakpoint site. Therefore, using
a combination of both of these techniques, one can create any mutation or genetic change
at any part of any DNA. These very recent discoveries has opened up the possibility for a
more indirect route in bringing back extinct species of animal. By comparing the genetic
structure of a species that is extinct to its most closely related relative, we can see often the
very small percentage of genes that have been altered over evolutionary time. By using
CRISPR techniques alongside HDR, we can isolate the differing genes in question of living
species in the zygote of any animal (e.g. an Asian elephant) and simultaneously reprogram
the genome to take on the phenotype of an extinct animal (e.g. a wooly mammoth). Allow
gestation to take place and upon birth, we have a genetically engineered extinct animal.
This brand new technique that is barely a year old has revolutionized genetic engineering
dramatically, and the possibilities beyond just this topic are incredibly far reaching; from
eradicating diseases such a malaria to breaking down antibiotic resistances which is
becoming an ever increasing problem. This technique is particularly useful if the DNA
sample from the extinct species has become fragmented over time and would not be
suitable for direct transfer through a cloning process or a PGC manipulation. However, the
question remains should a genetically engineered mammoth really be considered a
mammoth, even if the majority of genetic differences between it and the Asian elephant
were altered? Indeed artificially creating new life based of a deceased model could really be
referred to as true de-extinction is a matter of debate in many circles.
Cloning Techniques
From practically brand new technologies in CRISPR to a technique that has remained
relatively unchanged since its invention, the practice of cloning is still very much a
developing science even now. From the first uncontested mammalian clones of lambs
conducted by (Willadsen 1986) by the process of nuclear transfer, this relatively simple
concept has remained more or less the same when used in the present day. The most
famous case of successful cloning came in the form of Dolly the sheep, produced by (Wilmut
1998) for the Royal Institute in Edinburgh, Scotland. In this classic experiment of the form,
the donor culture of cells was derived from mammary tissue, and was initially deprived on
nutrients to slow down cellular processes. Then a Scottish Blackface ewe provided an oocyte
that was removed during metaphase II. The spindle fibres and chromosomes were removed
using a glass micropipette and the donor cell was placed adjacent to the now enucleated
oocyte. An electrical stimulus was then presented to the two cells, which initialised the
fusion of the oocyte and the donor cell. From there, the oocyte was fertilised, placed back in
the mother’s uterus and gestation was allowed to occur. From there, Dolly was born, to
which allelic DNA microsatellite analysis confirmed that Dolly belonged to the same Finn
Dorset breed of sheep from which the donor cell derived from. Dolly was a reproductively
competent clone who went on to produce 3 offspring.
From the initial beginnings of Dolly also gave birth to the idea of transgenic cloning, which
was successfully achieved by (Schnieke, Kind et al. 1997), who successfully trans infected
foetal sheep fibroblasts in culture with the human gene for clotting factor IX, alongside a
regulatory gene to restrict its expression to the mammary gland, alongside a gene for
neomycin resistance to concentrate the culture of genetically modified fibroblast tissue.
These fibroblast tissues were then fused with an oocyte which gave birth to Polly the sheep,
who successfully produced human clotting factor in her milk, giving way to new advances in
increased drugs and other biological products in transgenic mammals over those initially
produced in bacteria.
As it has been proved several times after these initial experiments since then, it is become
an undeniable fact that transgenic mammals, and thusly all animals, can be successfully
engineered. On this account, should a cell from an extinct species be preserved to allow the
cellular fusion of itself with an enucleated oocyte of its closest common ancestor, then in
theory, it could give birth back to the species.
Following on from this idea, a variety of experiment have been conducted in the pursuit of
whether it would be possible to use somatic cell nuclear transfer (SCNT) and genomics to
bring back extinct species. These experiments include the successful birth of offspring using
SCNT using frozen mouse cells without a cryoprotectant (Li, Mombaerts 2008), which then
caused follow up experiment where cloned offspring were produced from ESCs from frozen
mice bodies (Wakayama, Ohta et al. 2008). The pinnacle of this initial investigation over
whether cloning was viable came from the successful transgenic mouse created which
contained Col2A1 enhancer gene from the already extinct Tasmanian tiger Thylacinus
cynocephalus) whose DNA has been preserved in ethanol for over 100 years (Pask,
Behringer et al. 2008). All these experiments give excellent evidence that as long as DNA has
been preserved in some form (in these cases, in cryostasis or in alcohol) then the DNA at
least can be resurrected in other animals. See Table 1 overleaf for a list of animals that are
on the brink of extinction or are extinct whose DNA samples would be good candidates for
trials for cloning conservation (Pina-Aguilar, Lopez-Saucedo et al. 2009).
After so many experiments in this field, the first successful cloning of an extinct animal was
achieved in the form of a Pyreanean ibex (Capra pyreaica pyrenaica) through SCNT
techniques from cells of the last living Ibex (Folch, Cocero et al. 2009). Skin biopsies of the
ibex were taken, expanded in vitro and preserved in liquid nitrogen. Using SCNT methods
outlined above, a female ibex was born from a domestic goat, and however it died within
minutes of being born due to severe respiratory distress. The post-mortem report showed
that the new-born had an atelectasis, as well as a lump on the left long, showing the cause
of death. However, nuclear DNA microsatellite analysis as well as mitochondrial DNA
analysis confirmed that the new-born did spawn from the last Pyrenean ibex.
The occurrence of severe developmental abnormalities in cloned animals is an unavoidable
observation when assessing the technique’s effectiveness. For a start, SCNT effectiveness
was assessed to be 4%, being the number of new-born animals in good health for every
reconstructed embryo, regardless of the species of origin (Brisville, Fecteau et al. 2011). In
Brisville’s study on respiratory illness in cloned cattle, 9 out of 18 calves developed
respiratory diseases within 12 hours of birth, whilst 6 developed respiratory illnesses 24
hours after birth with only 3 appearing healthy. On top of this, due to the nature of using
adult genetic information in the creation of new life, the length of telomeres on the
chromosomes are significantly shorter compared to a natural birth, irrevocably reducing the
lifespan of the cloned organism. Also, large offspring syndrome (LOS has also been observed
in cloned cattle and sheep as well as a variety of other defects across the board in regards to
cloning(Young, Sinclair et al. 1998). It is clear that in regards to regularly successful cloning
we are still very much far away from a solution that can avoid the myriad of problems of
using SCNT techniques in conservation and the resurrection of extinct species
Concluding Thoughts
It is apparent that over recent years that huge advances in de-extinction has occurred over
recent years from science fiction to a very apparent reality with attempts already being
made to bring back animals such as the ibex. However there is clearly still a significant
amount of fine tuning required to improve the possibilities of having successful
resurrections. In the case of PGCs, this is proving to be potentially the most reliable source
of conservation technique out of the 3 that this paper has covered, but has only really been
seen working with genetic material taken from living organisms, as well as the fact the
success rate in which successful fertilisation can occur is really very small to be used in
organisms that naturally produce less offspring; not to mention the complications of using
this type of technique on animals who require this process to occur in utero. CHIP/Cas9
technologies appear to be a highly promising new technique in multiple areas of medicine,
and definitely has the ability to artificially create life based on extinct species. However the
technique is still in its infancy as well as the fact that should extinct species be created via
this method, it could be argued that the end product of the experiment could only be
considered a genetically engineered version of its closest living relative and could not give a
true understanding of how the extinct species lived when it were alive. In terms of using
SCNT techniques, its success rates are also very low, with added issues in regards to the high
probability of developmental defects as well as their shortened lifespan due to shortened
telomeres. Is it right to bring back an animal (in which most cases, were killed due to human
involvement) to only expose it to a stunted life?
The generalised ethical issues regarding de-extinction are also vast. It is natural to feel
responsible for animals such as the American Passenger Pigeon, one of the most populous
birds in the world, were drove from the world by human interference, but naturally we will
then have to make sure we do not become blasé in regards to the conservation of species.
For those animals that died out simply due to natural selection, such as the mammoth, is it
really right that we bring back an animal in which its chances of survival were minimal even
before humans began to dominate the Earth and inevitably change the environment around
us to become possibly less inhabitable? Is there an ecological benefit for bringing back an
animal that could help better the planet? Or is it a simple matter of bio-objectification and
human curiosity that is driving us to defy nature at its most basic level.
All these questions need to be considered on top of improving the techniques in place
currently before we can really consider bringing back animals that have become extinct,
however the fact that an animal as legendary as the woolly mammoth possibly being alive
and well in this lifetime is something that is undeniably exciting and very, very possible.
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Acknowledgement
I would like to thank Ben Novak and his team at Revive and Restore for sending me several
articles on CRISPR techniques as well as the use of PGCs, which is the basis of their
continuing work to bring back the American Passenger Pigeon to the skies.