Artificial cloning of domestic animals

Carol L. Keefer1
Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742

Edited by Francisco J. Ayala, University of California, Irvine, CA, and approved April 15, 2015 (received for review February 6, 2015)

Domestic animals can be cloned using techniques such as embryo the blastomeres of an early embryo and forming two or more
splitting and nuclear transfer to produce genetically identical smaller embryos. Initial studies were performed to ask key
individuals. Although embryo splitting is limited to the production questions regarding control of lineage development: When is a
of only a few identical individuals, nuclear transfer of donor nuclei cell’s fate set and how plastic is that fate? Studies in amphibians,
into recipient oocytes, whose own nuclear DNA has been removed, rabbits, and mice suggested that the very early cleavage stages
can result in large numbers of identical individuals. Moreover, (two-cell to four-cell) were flexible and that each blastomere
clones can be produced using donor cells from sterile animals, such could yield a viable blastocyst. At later stages, the blastomeres
as steers and geldings, and, unlike their genetic source, these clones could no longer independently form a viable blastocyst due to the
are fertile. In reality, due to low efficiencies and the high costs of loss of mass as each blastomere underwent cleavage division.
cloning domestic species, only a limited number of identical in- That did not mean that the blastomere nucleus was incapable
dividuals are generally produced, and these clones are primarily of directing full development, but rather that it was unable to
used as breed stock. In addition to providing a means of rescuing stop the developmental clock and replace the lost mass before
and propagating valuable genetics, somatic cell nuclear transfer continuing. Prior to the blastocyst stage, cells in the early embryo,
(SCNT) research has contributed knowledge that has led to the called blastomeres, divide without increasing in mass between
direct reprogramming of cells (e.g., to induce pluripotent stem cells) each division: thus the term cleavage divisions—each cell cleaves
and a better understanding of epigenetic regulation during embry- in half. This constraint led to the obvious question: If you provide
onic development. In this review, I provide a broad overview of the additional mass, can later staged blastomeres—or more appro-
historical development of cloning in domestic animals, of its appli- priately—can the nucleus of a later staged blastomere direct
cation to the propagation of livestock and transgenic animal pro- complete development to the blastocyst stage and be capable of
duction, and of its scientific promise for advancing basic research. continued development resulting in a normal, live offspring? Pio-
neering studies in the 1950s and 1960s in frogs demonstrated that
SCNT | cloning | nuclear transfer | embryo | livestock nuclei from embryos up to the tadpole stage were capable of
directing normal development, resulting in adult individuals, but

T he word clone can mean different things to different people.

In molecular biology, it refers to the process of making
identical copies of DNA. In cell biology, it is the propagation of a
that nuclei from adult tissues were able to direct development only
to the tadpole stage (10, 11). Despite the failure to obtain adult
individuals after nuclear transfer of adult cells, the studies did
progenitor cell to obtain a population of genetically identical demonstrate the developmental plasticity of differentiated, somatic
cells whereas, in animal biology, cloning refers to the production cell nuclei. The unknown (at that time) regulatory mechanisms
of genetic copies of individual animals using nuclear transfer. controlling cell-specific gene expression could be reset back to
Advanced reproductive methods involving microsurgery, embryo the embryo stage.
culture, and transfer into recipients (surrogate mothers) are re- Systematic examinations of mammal embryonic plasticity
quired to produce animal clones (Fig. 1). More specifically, a could not be conducted until appropriate in vitro culture con-
nucleus from a cell of the donor individual is inserted into an ditions had been established in the 1960s and 1970s (12–14).
oocyte whose own nuclear DNA has been removed (enucleation). Subsequently, controversial studies in the 1970s suggested that
This reconstructed oocyte is activated to continue embryonic de- nuclei from cells that had undergone the first lineage differen-
velopment. Embryos resulting from this procedure can result in tiation (that is, cells that had formed the inner cell mass) could
the production of a live, genetically identical individual after direct normal development if substituted for the zygotic nucleus
transfer into a recipient, although at a relative low efficiency (15). However, failure of other research groups to replicate these
(Table 1). The fact that such a complex procedure works at all is studies led some scientist to state that mammalian nuclei after
amazing and is the result of decades of pioneering research. In embryonic gene activation were unable to direct development
this review, the historical work in domestic species leading up to due to irreversible programming changes (16). At this time, ad-
the development of somatic cell nuclear transfer (SCNT), along vances in reproductive technologies involving farm animals,
with the practical applications of this technology, will be dis- primarily sheep and cattle, allowed animal scientists to adapt
cussed. As illustrated below, basic research regarding the bi- such techniques as embryo splitting and blastomere cloning with
ological mechanisms of SCNT has led to scientific advances in the a focus on improving production efficiencies and genetic ad-
areas of reprogramming, cell fate determination, and epigenetic vancement, in addition to asking questions about developmental
plasticity. Because developmental biologists, focused on more
regulation during development. Moreover, as discussed in the
traditional models (e.g., mouse and frogs), tend not to read the
following sections, SCNT in domestic animals will continue to
more agricultural-related science journals, the advances made by
provide promising scientific and practical insights through its
application to transgenic and biomedical models.

Setting the Stage—Historical and Evolutionary Perspective This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sci-
ences, “In the Light of Evolution IX: Clonal Reproduction: Alternatives to Sex,” held January
How much have we diverged from nature’s method of cloning? 9–10, 2015, at the Arnold and Mabel Beckman Center of the National Academies of Sciences
Even omitting most other forms of plant and animal life and and Engineering in Irvine, CA. The complete program and video recordings of most present-
ations are available on the NAS website at www.nasonline.org/ILE_IX_Clonal_Reproduction.
focusing on vertebrates—animals with backbones—examples of
clones abound in nature. Identical twins are the obvious exam- Author contributions: C.L.K. wrote the paper.

ples, but perhaps more intriguing are armadillos, in which the The author declares no conflict of interest.
offspring in a litter are all clones derived from one zygote (9). The This article is a PNAS Direct Submission.
simplest form of artificial cloning is embryo splitting—separating 1
Email: [email protected].

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compared with somatic cells (34). After fertilization, the sperm
DNA undergoes active demethylation, and the maternal DNA
undergoes passive demethylation. Some genes maintain a pa-
ternal or maternal imprint such that active transcription occurs
only from one parental chromosome; these imprinted genes of-
ten play critical roles in placentation: e.g., the imprinted growth
factor, IGF2, is expressed by the paternal and not the maternal
allele whereas the receptor, IGF2R, is maternally expressed (35).
Although these imprints are generally described by their DNA
methylation patterns, the complex mechanisms and levels of
imprinting, including histone modifications, are still being deci-
phered. Why is the imprint important during artificial cloning?
After all, the donor cells have both maternally and paternally
derived chromosome sets. During development, as cells undergo
lineage differentiation, epigenetic changes are continually being
established that alter the genetic program for cell-specific gene
expression. These changes include multiple levels of epigenetic
alterations, including DNA methylation and posttranslational
modifications of histone proteins (Fig. 3). During reprogram-
ming by the ooplasm, these patterns must be reset back to the
zygotic patterns. This requirement places a strain on the capa-
bilities of the reprogramming factors within the ooplasm as they
Fig. 1. Somatic cell nuclear transfer process. have evolved to reset the paternal and maternal gametic epige-
netic patterns, not that of a somatic cell. Therefore, it is not
surprising that oocytes are not able to fully and correctly re-
animal scientists were largely unrecognized until the production of program the somatic epigenome. In fact, it is surprising that they
Dolly merited publication in Nature (6). Although the fact that an are capable of achieving a sufficiently appropriate epigenome
adult nucleus could indeed direct normal development (resulting that allows full development. As remarked by other authors,
in a live offspring) was revolutionary for developmental biology, it nature allows a certain amount of flexibility in the epigenome
followed a series of discoveries that suggested such a possibility and gene expression during growth and development (28). As
(Fig. 2). might be anticipated, clones that fail during gestation and/or
The initial attempts to artificially clone domestic animals in- have physiological abnormalities have been found to have ab-
volved embryo splitting. Steen Willadsen demonstrated that normal epigenetic patterns whereas those that thrive have a
twins could be produced in sheep (17) and cattle (18) after comparatively normal pattern (28, 36). It is also interesting to
splitting of cleavage-staged embryos and transfer of the demi- note that the gestational losses and abnormalities observed in
embryos into recipients. These studies demonstrated that triplets SCNT were also noted during the development of in vitro em-
and even quadruplets could be obtained, albeit at lower fre- bryo production and culture techniques in domestic species in the
quencies due to the loss of cellular mass. To overcome this 1990s. In sheep and cattle, affected offspring were typically larger
limitation, Willadsen used a modified version of the nuclear

DEVELOPMENTAL
than normal; thus, the term “large offspring syndrome” was coined
transfer technique that had been used in the earlier amphibian

BIOLOGY
(37). It was determined that exposure to serum and coculture al-
cloning studies described above (Fig. 1). In brief, the oocyte’s tered embryonic epigenetic methylation patterns (38, 39). With
DNA was removed by aspirating the portion of ooplasm con- improvements in culture media, the incidence of large offspring
taining the chromosomes, thus forming a cytoplast; the donor after in vitro embryo production no longer seems to be the issue it
cell was placed abutting the ooplasmic membrane; and the once was although the more subtle epigenetic changes that may have
cytoplast and donor cell were fused together. Using this procedure, long-term consequences on offspring health are of great interest. A
Willadsen obtained live lambs (19) and calves (20) after transfer of number of research groups are exploring these subtle epigenetic
the reconstructed embryos into surrogates. Subsequently, several changes that can occur during gamete, embryo, and early pregnancy
other research groups with ties to the agricultural industry began with potentially long-term consequences on offspring (40, 41).
exploring the possibilities of embryo and embryonic cell nuclear
transfer, achieving success with progressively later stages of em- Propagation of Genetics
bryos (Table 2). In 1996, researchers at the Roslin Research In- Once lambs and calves had been produced using embryonic and
stitute reported successful production of live lambs using long-term fetal cells, publications dealing with SCNT research escalated
cultured embryonic (21) and even transgenic (22) cells. These
achievements were soon followed by the report of the production
of a lamb (Dolly) using cultured somatic cells that had been Table 1. Cloning efficiencies in domestic livestock
obtained from an adult (6). Although much has been made of the Efficiency per Efficiency per
low efficiency of somatic cell nuclear transfer (SCNT)—Dolly was Species reconstructed oocyte, % transferred SCNT embryo, % Refs.
the single live offspring that resulted from 29 transferred recon-
structed embryos for which 247 oocytes had been manipulated— Cattle 1.7 11.5 1
the fact that a live lamb was produced is still astounding. Goat 6 6 2
To fully comprehend the barriers to artificial cloning, one Horse 0.8 19 3
must first understand the processes of gametogenesis and fer- Pig 0.3 5–13 4, 5
tilization. During mammalian development, the primordial germ Sheep 0.3 3.4–5.9 6–8
cells in the developing fetus migrate to the gonadal ridges and, Estimates based on selected publications that provide sufficient datasets
depending on the sex of the fetus, form either oogonia or sper- to determine cloning efficiencies based both on a per reconstructed oocyte
matogonia. DNA methylation patterns are established such that and a transferred embryo basis. Transferred embryos are often highly
sperm are hypermethylated and oocytes are hypomethylated selected and can represent a very small subset of SCNT embryos produced.

Keefer PNAS | July 21, 2015 | vol. 112 | no. 29 | 8875

 a gaur nucleus into a bovine oocyte died shortly after birth (47)
although more recent attempts seem to have been more successful
(48). Success has also been achieved between endangered cats
using oocytes from domestic cat as recipient (46). Studies suggest
that the frequent failures of interspecies SCNT are due to a
number of factors including incomplete activation of the embry-
onic genome and nuclear–mitochondrial incompatibilities (49).
SCNT and Transgenic Animal Production
SCNT may provide the most advantages for the production of
transgenic animals. Although transgenic animal production is an
efficient process in mice in which multiple methods can be used,
including pronuclear microinjection of DNA constructs, chimera
production using transfected embryonic stem cells (ESCs), or
SCNT using transgenic donor cells, only the last method has any
Fig. 2. Timeline of key points during development of SCNT in domestic practical application in domestic species. Pronuclear injection
livestock (6, 17–33).
has resulted in transgenic pigs, sheep, goats, and cattle, but at
much lower efficiencies than mice and at much greater costs (50,
from a few studies a year to hundreds. At this time, more than 51). SCNT allows production of transgenic offspring after se-
twenty different species have been cloned by SCNT techniques lection and characterization of donor cells. This process ensures
although not all offspring have survived long-term. Of particular that the offspring are transgenic and have the appropriate
interest were the advantages that embryo cloning and SCNT number of copies of the transgene, and virtually ensures that the
offered for the propagation of valued genomes, whether for animal contains and expresses the transgene. Transgenic cattle,
animal production purposes or rescue of rare genotypes. Animal goats, pigs, and sheep have been produced that express industrial
genetics companies that sell semen and embryos for genetic proteins (e.g., spider silk), biopharmaceuticals (e.g., antithrom-
improvement of dairy and beef herds could take advantage of bin), and human polyclonal antibodies (51). Moreover, animals
SCNT to expand their products. Clones of valued dams and sires have been produced with modified production traits, including
can be produced by SCNT, thus extending their reproductive increased casein in the milk (52), altered fatty acid composition
output potential. SCNT can also be used to propagate hybrids, to (53), and resistance to mastitis (54). Prion protein, the causative
increase the speed of genetic gain through selection of animals agent in mad cow disease, has been knocked out in cattle using
with superior phenotype and genotype, and even to replicate SCNT (55). Despite advantages conferred by these transgenic
animals with advantageous genotypes whether or not that animal modifications to production traits or disease resistance, none of
is fertile (e.g., steers and geldings). Examples include the cloning these animals will be used for food production due to regulatory
of a prized Texas Longhorn steer (42) and racing mules (43). roadblocks (56, 57). Thus far, only one product from a transgenic
Moreover, animals that have died can be cloned as long as viable animal has been approved in the United States and Europe for
cell samples were collected and stored. A resurrected prized bull, human use, and that is the biotherapeutic protein antithrombin
Starbuck II, produced daughters that had normal chromosome (ATryn). Whether transgenic animals ever fulfill the animal
stability (telomere lengths) and hematological, physiological, and production-related promise researchers envisaged will depend
reproductive parameters (44) although his semen was never sold on societal acceptance and revised regulatory guidelines. More
commercially due to Canadian governmental regulations. In likely is progress in establishing medical models of human and
animal disease for biomedical research. SCNT in domestic ani-
Texas, beef cattle have been resurrected based on their carcass
mals has been used to study the potential of regenerative med-
traits (45). Unlike the steer from which the carcass was obtained,
icine. For example, cloned pigs have served as both controls and
these cloned calves can look forward to siring offspring that are
recipients for neural stem cells, demonstrating the potential for
expected to have superior meat production traits.
In addition to propagation and/or replication of domestic species, spinal cord repair (58). SCNT has also been used to produce
SCNT has been used to propagate genetics of endangered species medical models for cystic fibrosis, diabetes, retinitis pigmentosa,
with mixed success because often this process involves interspecies cancer, amyotrophic lateral sclerosis, and other diseases (58–60).
SCNT (46). In fact, most efforts involving interspecies transfers, in These newly developed models hold great promise for providing
which an oocyte from one species is used as the recipient for a insight into diseases and should lead to new therapeutic treatments.
nucleus from another species, have not been successful. Only a few
cases of interspecies SCNT between closely related species have
resulted in the actual production of offspring. Frequently, these
animals do not thrive and die relatively soon after birth. As a case
in point, the first gaur calf that was produced by transplantation of

Table 2. Use of progressively more advances staged nuclei for

SCNT in cattle
Efficiency per
Stage of donor nuclei transferred SCNT embryo, % Refs.

Morula (16–64 cell) 10–25 23–25

Recloned morula 2–4 23, 24
Inner cell mass (ICM) 13–15 26, 27
Embryonic/fetal 5–15 28–31 Fig. 3. Epigenetic factors regulate DNA availability to transcriptional ma-
chinery (transcription factors, polymerases, etc.) and are involved in the
Adult 5–15 28, 29, 32, 33
control of cell tissue-specific gene expression.

8876 | www.pnas.org/cgi/doi/10.1073/pnas.1501718112 Keefer

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Basic Research Questions Owing to time and cost commitments, only a few studies have
SCNT is also being used to answer basic questions in developmental looked at the long-term consequences of cloning on such phys-
and reproductive biology. The majority of publications on SCNT iological parameters as reproductive performance or health. A
describe efforts to overcome the inefficiencies of the process itself. large-scale project involving 96 cow clones and 40 corresponding
These reports include attempts to identify the best source and genetic donors, as comparative controls, was carried out over a
treatment of donor cells, improved oocyte activation protocols, and 6-y period. In this longitudinal study, Polejaeva et al. (65) de-
methods of chemically assisted reprogramming. In the latter case, termined that the ability of clones to produce transferrable-
either donor cells before SCNT or the reconstructed embryo after- quality embryos after artificial insemination or in vitro embryo
ward are exposed to chemicals thought to either stimulate or inhibit production was not different from that of their genetic com-
various enzymes involved in remodeling chromatin. Many of these paratives that had been produced through normal breeding
publications report conflicting findings, and rarely are a sufficient practices. Other work has focused on the health of clones: these
number of embryo transfers performed to establish credible evidence studies suggest that clones that survive the critical neonatal pe-
of any real improvements in efficiencies. Nevertheless, chemically riod are generally normal physiologically. Cattle clones surviving
assisted reprogramming during SCNT does hold promise (61). greater than 200 d were found to be essentially equivalent in terms
Due to the cost and extended generation times for domestic of animal health and milk and meat production performance as
species, most of these studies, so far, have focused on early em- conventionally bred cattle (66). These studies support the fea-
bryonic and fetal development. As with epigenetic patterns de- sibility of using clones in various comparative studies. For ex-
scribed above, many of these studies report either the normal ample, multiple copies of genetically identical embryos produced
expression of key genes after a reconstructed embryo passes a by SCNT can be frozen and subsequently transferred at pre-
developmental checkpoint (e.g., blastocyst formation) or abnor- determined intervals, resulting in genetically identical individuals of
mal gene expression after failure to pass such checkpoints (28, 36). different ages. This approach was used in a recent publication in
These experiments focused mainly on how SCNT worked or did which the method of ovarian stimulation, but not maternal age,
not work, and few use SCNT to explore questions regarding de- was found to be associated with lower mitochondrial copy
velopmental regulation of genes. One exception includes the use of number in oocytes obtained from genetically identical cow clones
SCNT to explore the differential regulation of POU5f1 (OCT4), a of different ages (67). Future studies taking advantage of such
key transcription factor involved in trophectoderm formation, in unique research opportunities provided by SCNT may help an-
bovine and mouse blastocysts (62). This study provided insight into swer questions and solve technical issues in reproductive medi-
species-specific gene regulation during early development that cine and regenerative medicine.
would not have been achievable without SCNT.
In a limited number of cases, SCNT has been used to test Conclusion
hypotheses that could not be easily answered through other In general, SCNT efficiencies have improved only marginally
methods. For example, reproductive immunologists had long over the past decade, with the generally accepted rate of 5–15%
questioned the role of foreign paternal antigens in the estab- of transferred embryos resulting in live offspring (28). Direct
lishment and maintenance of pregnancy. Cesare Galli et al. used comparisons of efficiencies reported by various research groups
SCNT to investigate this question. Cultured somatic cells from a are often difficult because only subsets of embryos may have
mare were used as nuclear donors in SCNT. Two of the resulting been transferred or reported. Strict selection of embryos for
cloned embryos were then transferred into the same mare, transfer can result in improved pregnancy rates, per cloned
resulting in the establishment of a full-term pregnancy and the embryo transferred, that do not reflect the true viability of the
birth of a live foal (63). This birth demonstrated that a mare could full set of reconstructed embryos. Nevertheless, SCNT research

DEVELOPMENTAL
successfully carry a pregnancy initiated by her own identical clone, has contributed knowledge that has led to the direct reprogram-

BIOLOGY
which implied that foreign paternal antigens are not necessary for ming of cells (e.g., inducing pluripotent stem cells) and to better
establishing a viable, full-term pregnancy (63). Additional studies understanding of epigenetic regulation during embryonic de-
using SCNT could help further decipher the role of paternal anti- velopment and has provided means of propagating and rescuing
gens during pregnancy (64). valuable genetics and establishing large-animal biomedical models.

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