Conservation Genetics Resources (2021) 13:349–357

https://doi.org/10.1007/s12686-021-01204-9

TECHNICAL REVIEW

Conservation of endangered species through somatic cell nuclear

transfer (SCNT)
Ambreen Iqbal1   · Jiang Ping1 · Shaokat Ali1 · Gao Zhen1 · Jin Zi Kang1 · Pan Zi Yi1 · Lu Huixian1 · Zhao Zhihui1

Received: 4 April 2020 / Accepted: 8 March 2021 / Published online: 23 March 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Currently, the conservation of biodiversity is a pressing issue due to the extinction and endangerment of many species.
There are multiple reasons for the extinction or endangerment of species including anthropogenic (poaching, hunting,
and deforestation) and natural causes (forest fire, invasion of non-local species, climate change, and global warming). The
somatic cell nuclear transfer (SCNT) is an emerging tactic, which requires the nucleus from a donor somatic cell, which is
transferred to the enucleated oocyte in vitro, for the conservation of biodiversity, especially related to the wildlife which is
gradually decreasing with the fear of extinction. Somatic cell nuclear transfer has a broad spectrum of potential applications,
including the rescue of endangered species, the resurrection of extinct species, the production of transgenic animals, drug
production, and regenerative medicine. Reproductive cloning of animals by SCNT can also offer permanent gene editing,
genetic modification, and gene derives for the production of species adaptive to their native habitats and the destruction of
unwanted invasive species. The overall potential of this technique has not been explored yet, probably due to its limitations.
In this review, we discussed cloning through SCNT, the preservation of endangered species through SCNT, and the use of
modern genetical concepts in SCNT for the preservation of biodiversity.

Keywords  Endangered species · Genetic engineering · Somatic cells · Wildlife

Introduction The invasiveness through non-local species is also among

According to the International Union for Conservation of
Nature  (IUCN), an endangered (EN) species is the one
that is jeopardized to be extinct shortly. According to the
2020–3 IUCN red list version, about 128,918 species have
been assessed so far and from these more than 35,500 spe-
cies are threatened with extinction (https://​www.​iucnr​edlist.​
org/). The recently estimated extinction rate is 0.1 out of
every one million species per year (De Vos et al. 2015).
The ever increasing human population which associates
with growing demands for food and shelter culminates in
deforestation, overhunting, overfishing, increasing environ-
mental pollution, global warming in addition to the spread
of invasive species are leading causes for exploiting the eco-
system causing the extinction of many species (Hill 2014).
* Zhao Zhihui
zhzhao@gdou.edu.cn
1
Department of Animal Breeding and Genetics, Coastal
College of Agricultural Sciences, Guangdong Ocean
University, Zhanjiang 524088, Guangdong, China
the leading causes of endangerment and extinction of spe-
cies. Non-native rodents alone are the reason for about 37%
of the world’s threatened species (Jones et al. 2016). Further,
industrial booming and the use of pesticides causes the rapid
extinction of living beings through their respective harm-
ful residuals and toxicological effects. One such example
includes the accumulation of persistent organic pollutants to
hazardous levels which can disturb the normal cycling of the
food chain (Briggs and King 1952). Albeit, anthropogenic
activities are the major cause of biodiversity loss (Díaz et al.
2019) along with other subsidizing factors such as loss of
genetic diversity, mutation accumulation, inbreeding depres-
sion (Petrova et al. 2016), and habitat loss due to climate
change (Skogen et al. 2018; Horvath et al. 2019).
Cloning is used to produce the genetic replicas of an
individual. The most used application method of cloning
is SCNT which is one of the advanced biotechnological
tools of micromanipulation where a somatic cell nucleus is
transferred into enucleated oocyte cytoplasm. The mature
oocyte is reprogramed with the somatic cell’s nucleus and
the activation of mitosis starts after induction of electric

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shock. Next, the blastocyst stage is developed which is
responsible for making the embryonic stem cells by using
the inner cell mass of the blastocyst (Moo-Young and Bor-
dignon 2019; Wilmut et al. 2015). After completing the
embryonic stages, the blastocyst forms the fully mature
individual with the same genetic makeup as the donor of
the somatic cell nucleus (Willadsen 1986a). Dolly sheep
was the first cloned animal to be produced through SCNT
(Campbell et al. 1996).
SCNT, an advanced biotechnological tool in which a
somatic cell nucleus is transferred into enucleated oocyte
cytoplasm, can be used in different aspects including the
cloning of farm animals with desirable phenotypes (Bjorn
2008), conservation of endangered species, the revival of
extinct species through SCNT or iSCNT (Loi et al. 2007;
Gómez et al. 2009; Mastromonaco and King 2007; Wakay-
ama et al. 2008), and production of transgenic animals for
biomedical applications (Bordignon 2017).
The need for conservation of biodiversity is more than
ever before and it’s time to comprehend the gravity of the
situation and to act for saving the biodiversity on this planet.
This review will provide information about the conservation
of endangered species through SCNT in addition to the use
of genetic modification through SCNT and its application in
genetic engineering.
Breakthrough studies related to cloning
Before using SCNT in animal clone production, it was first
used for the production of the frog embryo through nuclear
transplantation in 1952 (Briggs and King 1952). After the
successful production of the frog embryo, the embryos
from different species were cloned including the mouse
(Illmensee and Hoppe 1981), rabbit (Stice and Robl 1988),
sheep (Willadsen 1986a, b), and cattle (Prather et al. 1987).
Despite the production of cloned embryos in the 80 s of the
twentieth century, production of the cloned animals was a
dream and scientists were ambiguous about the production
of clone animals by using SCNT until the creation of dolly
sheep in 1996 (Campbell et al. 1996). This revolutionary
production of the cloned sheep had negative feedback, but
this milestone achievement opened the gates of new research
in biotechnology. Following the production of dolly sheep,
the SCNT was applied for different genetic achievements
and many animals were cloned by using this revolutionary
technique. These achievements include chimeric animal
production (Cibelli et al. 1998), transgenic animal produc-
tion, and nuclear transfer (Harrison et al. 2002). Recently,
through the use of SCNT offspring of many species have
been produced such as sheep (Deng et al. 2013), goats (Zhou
et al. 2013), cattle (Luo et al. 2015), pigs (Ma et al. 2016;
Kwon et al. 2017), buffalo (Yang et al. 2010; Lu et al. 2018),
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Conservation Genetics Resources (2021) 13:349–357
and horses (Olivera et al. 2018). With time, the paradigm of
SCNT was exploited by researchers in ways other than just
the production of cloned animals (Yin et al. 2008; Guo et al.
2017). For instance, the SCNT technique helped to provide
innovative therapy for eliminating the risk of stem cell can-
cer (Hu and Fu 2012). SCNT is not only used to preserve
the threatened species but this revolutionary technique is
also used to improve milk and meat production, decline the
emission of greenhouse gases, and to produce therapeutic
proteins and cultured meat (Niemann et al. 2011).
SCNT has brought milestone achievements in the pro-
duction of meat, milk, medicine, and in the preservation of
endangered species besides that, it is also used to preserve
the elite dogs (Stafford 2006; Walsh and Emerita 2009). The
most distinguishing characteristic of the dogs is their smell
sense which is 10000–100,000 times stronger than humans
(Walker et al. 2003) making them better than the advanced
odor detecting machines. Dogs are also used to detect explo-
sives, drugs, and to assist blind people (Maejima et al. 2007;
Walsh and Emerita 2009). It is a costly and difficult task to
train the individual dog rather than preserving the attributes
of elite dogs through SCNT. SCNT was also used in canine
to improve the breed characteristics and the success story
was the production of snuppy, the first cloned dog (Lee et al.
2005). Further, a retired veteran rescue dog named Baekdu
in Korea performed the task of life-saving in the world for
about 6 years. Thus, SCNT was used to preserve the Baekdu
attributes’ for the production of threatened dogs (Kim et al.
2007b; Oh et al. 2008; Choi et al. 2016), pet dogs (Jang
et al. 2008; Park et al. 2011), top working-class dogs (Choi
et al. 2014), and transgenic dogs (Hong et al. 2009; Oh et al.
2011).
Somatic cell banks for conservation
programs in zoos
Biobanks are repositories used for the long term preservation
of biological samples for research purposes. Based on its
capacity, this idea can be handy in developing the bioreposi-
tories of endangered species in an attempt to save them from
extinction or to recreate them after extinction through SCNT.
Even, it can be used to preserve the genetic potential of elite
class animals to be used in a different time frame for their
better adaptability to the environment. For long term pres-
ervation, the somatic cells of threatened species are needed
to be saved in a biobank of wildlife (Comizzoli 2017). Many
of the somatic tissues of the wildlife have been preserved
with the cooperation of zoos (Verma et al. 2012; Wittayarat
et al. 2013; Yelisetti et al. 2016). Preserved somatic cells
of threatened wild felids gathered from different areas of
the world (León-Quinto et al. 2014). Two methods are used
in wild fields for the collection of the somatic cells, one is
Conservation Genetics Resources (2021) 13:349–357 351
the biopsy (Verma et al. 2012) and another one is necropsy
(Moulavi et al. 2017). Tissues are mainly collected from
different parts of the animal body including muscle (Thong-
phakdee et al. 2006), bone marrow, intestine, oral mucosa,
spinal cord (León-Quinto et al. 2014), abdomen (Wittayarat
et al. 2013), inner groin area (Mestre-Citrinovitz et al. 2016),
and ear skin (Praxedes et al. 2019; Santos et al. 2016) of
both genders (Thongphakdee et al. 2010; Wittayarat et al.
2013) at different ages (Hsahem et al. 2007b). Mostly, ear
skin is used for sample collection due to its ease of collection
(Changjun et al. 2011). A somatic cell biobank was devel-
oped at Buenos Aires Zoo in Argentina, for the collection
and culture of Jaguar’s somatic tissues (Mestre-Citrinovitz
et al. 2016). This biobank conducted the collection and pres-
ervation of 570 samples from 45 autochthonous threatened
species of the jaguar in a cryobank. Further, the genome
of two cat species, both genders, a flat-headed (Prionailu-
rus planiceps) and a marbled cat (Pardofelis marmorata),
preserved in 2003 in genome resource bank with the coop-
eration of the Zoological Park Organization. By using the
somatic cells from the cryobank, numbers of the endangered
species conserved including Iberian Lynx (Lynx pardinus) in
Europe (León-Quinto et al. 2011), Leopard Panthera uncia)
in Central Asia (Verma et al. 2012), Asian golden cat in
Southeast Asia (Wittayarat et al. 2013), Cheetah (Ancinonyx
jabutus) in Africa and Southwest Asia (Moro et al. 2015),
Jaguar (Panthera onca) in America (Mestre-Citrinovitz et al.
2016) and Asian cheetah (Ancinonyx jabutus vanticus) in
Africa and Southwest Asia (Moulavi et al. 2017).
Moreover, the somatic cells present in cryobanks can be
used to preserve the endangered species by using the tech-
nique of interspecies somatic cell nuclear transfer (iSCNT,
discussed in the next section) (Loi et al. 2011). By using the
nuclei of African wild cat and cytoplasm of the domestic
cat through iSCNT the first interspecies cloned cat was born
in 2004 (Gómez et al. 2004, 2008). The preserved somatic
cells were taken from epithelial and muscular tissues of
both cat species to produce another interspecies cloned cat
(Thongphakdee et al. 2010). The idea of cryopreservation
of biological tissues for saving the endangered species was
not remained limited to the somatic cell’s preservation but,
the semen of wild species was also saved in biorepositories
for the conservation of biodiversity (Holt et al. 2014). With
advancements in this technique, there is steady progress in
semen banking and cryopreservation (Comizzolli and Holt
2014). The establishment of the wildlife genome resource
banks (GRBs) for the collection of the living biomaterials
including DNA, somatic cells, germ plasma, and embryo
are fundamental components that are more used in research
(Comizzolli and Holt 2014). The black-footed ferret, a more
endangered species of North America, preserved through
natural and artificial insemination by using frozen semen
preserved up to two decades (Howard and Wildt 2009).
Interspecies cloning—a step
towards conservation of endangered species
In interspecies somatic cell nuclear transfer (iSCNT) the
nucleus of a donor organism is transferred into a recipient
enucleated oocyte of another species/family/order/class.
The early embryo is transferred to the surrogate mother
for development to term (Loi et al. 2011). The major use
of iSCNT is in the cloning of endangered species for their
conservation or de-extinction of extinct species for their
resurrection and evolutionary studies. The limited avail-
ability of oocytes from extinct, endangered, or wild spe-
cies makes the dependency on other ooplasm of different
species (Gómez et al. 2009).
There are two interspecies cloning techniques:
• Interspecies somatic cell nuclear transfer (iSCNT)
• Interspecies handmade cloning (iHMC)
In iSCNT, the enucleation of the oocyte is done by
keeping zonapellucida intact on a micromanipulator while
in iHMC there is no need for a micromanipulator because
all of the procedures are performed by hand. The success
of the complete development of an embryo through iSCNT
depends on several factors including the cell cycle stage,
the genotype of donor cells, oocyte cytoplasmic source,
and synchronization (Beigh et al. 2017). The success of
the iSCNT will increase if the two species have a close
relationship while with the increase in the divergence
between the species, the success rate of iSCNT decreases.
The iSCNT among the donor and recipient with a simi-
lar type of placenta, gestation period, and reproductive
physiology can have a higher success rate. Although the
cloning of closely related species is possible, the success
rate is very low even after the successful implantation of
the live embryo (Wani et al. 2010). There are many fac-
tors responsible for the death of the embryo, for example,
mitochondrial unsustainability, inappropriate demethyla-
tion of the donor genome, and incapability of maternally
inherited factors.
Different endangered animals have been cloned by
iSCNT including mouflon (Loi et  al. 2001), banteng
(Sansinena et al. 2005), the grey wolf (Kim et al. 2007a),
gaur (Lanza et al. 2000), gaur calf (Srirattana et al. 2012),
coyotes (Hwang et al. 2012) cheetah (Moro et al. 2015),
tiger, lion, and leopard (Yelisetti et al. 2016). Notably, dif-
ferent studies have embarked on the production of moru-
lae and blastocysts through interspecies cloning between
two very distant taxonomically related organisms. These
include interfamily iSCNT between bovine and pig (Uhm
et al. 2007), inter order iSCNT of cat and panda to rabbit
(Wen et al. 2005) camel and Tibetan antelope to rabbit
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352
(Zhao et al. 2006), human to rabbit (Shi et al. 2008), dog
pig (Sugimura et al. 2009), tiger to a pig (Hsahem et al.
2007a), human to bovine (Illmensee et al. 2006), human to
ovine (Hosseini et al. 2012), and interclass iSCNT between
chicken and rabbit (Liu et al. 2004).
One recent example of iSCNT is the interspecies cloning
of Bactrian and dromedary camels having similar reproduc-
tive physiology and diffused epitheliochorial placentation
(Wooding et al. 2003). Both sub-species have an almost same
gestation period of about 374–419 days in Bactrian camels
while in dromedary camels it is about 365–410 days. The
oocytes from the dromedary camel were used as a source
of cytoplasm, while the skin cells taken by biopsy from the
Bactrian camel were the source of the nucleus. Dromedary
calf was born healthy and lived for 7 days after that the calf
was died because of E. coli infection. This breakthrough
study has ignited the importance of interspecies cloning for
the preservation of Bactrian camels which is the 8th most
endangered large mammal.
The Asiatic cheetah (Acinonyx jubatus venaticus) is a
critically endangered subspecies by IUCN red-list because
of the habitat, road accidents, persecution killings, poaching
(Farhadinia et al. 2012), and road accidents which account
for 40% of deaths (Farhadinia et al. 2013). In 2010, a female
cheetah died in a road accident in Yazd protected area, Iran.
The somatic cells from the frozen body, at − 20 °C were used
as a source of the nucleus for iSCNT with in vitro matured
cat oocytes as the cytoplasmic recipient. However, when the
genetic material from non-viable nuclei of an Asiatic chee-
tah was transferred to the enucleated cat oocyte, all of the
cleaved embryos were unable to reach the blastocyst stage.
The probable reason for the early embryonic arrest of iSCNT
embryos was the mismatch of mtDNA between the donor
cell and recipient oocyte (Sansinena et al. 2011). Contrarily,
when live proliferating cheetah cells were used for iSCNT,
about 16.7% of embryos developed into the blastocyst stage
(Moro et al. 2015). This study is evidence that the taxonomi-
cal relationship between a cheetah and the domestic cat is
close enough for the development of iSCNT embryos to at
least the blastocyst stage. Moreover, in a recent study, Yeli-
setti et al. (2016) used iSCNT for rabbit enucleated oocytes
and skin biopsied cells of leopard, lion, and tiger. Remark-
ably, the percentage of fused couplets was increased and the
development of blastocyst was enhanced after the treatment
of donor cells with sodium butyrate which is an inhibitor
of histone deacetylase enzymes. The results of this study
suggest that the use of epigenetic modification can improve
the chances and development of preimplantation iSCNT
embryos between wide taxonomically related matches.
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Gene editing as a tool for conservation
Since the discovery of CRISPR-Cas9, it has been much pre-
cise, easier and cheaper to genetically modify the organisms
as compared to previously used tools such as transcription
activator-like effector nucleases and zinc-finger nucleases
(Du et al. 2016). This genetic tool can be adjoined to the
SCNT for genetic modification, gene editing, and gene
drives. Though, using CRISPR-Cas9 does not warrant the
avoiding of unforeseen and unwanted outcomes of traits con-
trolled by multiple genes. Still, this revolutionary genetic
tool can be applied in the conservation of biodiversity
through exploration and modification of genes involved in
disease resistance, resilience to climate changes, and for a
better adaptation of species to the local environment (Weeks
et al. 2016; Reardon and Sara 2016).
Further, all progeny of the gene-edited parent can have
both copies of the edited gene through CRISPR-based gene
drives. This could help in the spread of deleterious genes in
harmful invasive species for enhancing the survival of local
threatened species (Esvelt et al. 2014; Webber et al. 2015).
The likelihood of genetic modification in mosquitoes for the
prevention of diseases spread by mosquitoes is an excellent
example (Reardon and Sara 2016).
The generation gap is the main deriving factor in gene
derives because it is cheaper and efficient in species with
short generation times as compare to long-lived species. The
reversing of gene derives is a difficult task but it is being
exploited for use (Reardon and Sara 2016) and the deleteri-
ous genes could spread to the natural species leading to their
global extinction (Webber et al. 2015). Therefore their first
uses are to take on targeting the invasive species in isolated
islands (Webber et al. 2015). Alongside its uses in introduc-
ing the deleterious genes in harmful and unwanted species,
gene drives can be used in the conservation of wanted spe-
cies. One such example is the conservation of threatened
species by increasing resistance to biological or abiotic
threats (Esvelt et al. 2014). Moreover, genetic engineering
for the development of iPSCs could be used to develop the
donor nucleus of an extinct, endangered, or wild species.
Endangered species including, drill (Mandrillus leucophaeu)
and northern white rhinoceros (Ceratotherium simum cot-
toni) were generated by iSCNT by using induced pluripotent
stem cells (iPSCs) (Ben-Nun et al. 2011).
Identifying adaptive potential
for conservation
Adaptation to the environment is a necessity for the
survival of a species. With advancements in genetics,
genomic and transcriptomic data is used to identify the
Conservation Genetics Resources (2021) 13:349–357 353
adaptation-related loci. With the use of this strategy,
through gene editing of genes responsible for adaptation,
in SCNT programs, the conservation of genetic diversity
can be taken to another level of adaptation and survival.
The identification of conservation units and reintroduction
programs could be fulfilled through this approach (Mcma-
hon et al. 2014; Shafer et al. 2015; He et al. 2016) with
the further genetic rescue of isolated, inbred populations
(Whiteley et al. 2015), and spotting of climate-resilient
populations (Christmas et al. 2016).
A large amount of genome-wide sequencing data for
multiple species is available which could be analyzed sta-
tistically for the identification of environmentally corre-
lated targets (Vitti et al. 2013; Whiteley et al. 2015). The
precise identification of targets is difficult in the case of
multilocus combinations, but in the case of single nucleo-
tide polymorphisms (SNPs), the genome-wide association
studies (GWAS) are conducted for links between traits and
environmental variables (Williams et al. 2014; Hoffmann
et al. 2015; Vitti et al. 2013). The statistical simulations
are just the hypothesis that needs to be validated through
evidence. In such kind of a recent study the SNPs cor-
related with climate gradients were identified in valley
oak in California (Sork et al. 2016). The utmost need for
conservation is the identification of populations with the
capacity to resist and adapt to the novel threats of climate
change (Harrisson et al. 2014). In case, these threats are
understood to be present or occur in the future, the pos-
sibility of targeting the specific adaptive traits could be
explored. In a recent example, GWAS with transcriptomics
was used for identifying the markers with a strong asso-
ciation to resistance for a distressing fungal pathogen in
European ash, enabling the rapid recognition of genetically
diverse tolerant trees (Harper et al. 2016).
Limitations of SCNT and its uses
in conservation
Culture, religion, and ethical problems are the main hur-
dles for human cloning while animal cloning’s main hurdle
is its very low success rate which is less than 5% on aver-
age (Wilmut et al. 2015). Cloning through SCNT is an
inefficient process because for the creation of Dolly sheep,
the cloning of about 277 sheep was done which is about a
0.4% success rate. There are many abnormalities associ-
ated with SCNT such as early embryonic death, placental
abnormalities (Watanabe and Nagai 2009), growth abnor-
malities, and post-natal mortalities (Hill 2014).
Further, the use of this holy-grail in conservation biology
has many barriers including the lack of knowledge to the
practitioners as compared to the researchers which limits the
extension of this technique. Therefore, the proper guidance
of practitioners is necessary before using any new technol-
ogy. Additionally, the spread of gene-edited, gene-derived,
and de-extinct animals comes with the risk of disturbing the
local species or the environment. The ethical issues, global
and national laws, policies, rules, and regulations also act
as a checkpoint in the common usage of SCNT (Aslan et al.
2014).
Conclusion
The conservation of biodiversity is a major issue for bio-
logical researchers. Therefore, different aspects including
SCNT are under consideration of researchers for conser-
vation. SCNT has made major advancements in conserva-
tion through the reproductive cloning of many endangered
species and interspecies cloning of different animals is
underway. Many endangered species have been conserved
through benefiting this technique and the list continues. The
advanced concept of transgenic and genetically modified
organisms is a perk of having SCNT. But this biological
technique, like many others, comes with its limitations and
difficulties. Some of these include ethical and legal issues,
low success rates, high costs, and lack of awareness to the
practitioners. Therefore, further improvement and refine-
ment in the procedures of this revolutionary technology can
help in overcoming these limitations.
Declarations 
Conflict of interest  The authors do not have any conflict of interest.
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