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In 1996, when Dolly the sheep was born, a new, utopian era was expected to begin. Science fiction and popular culture instantly threatened the public with shortly upcoming human clones, portraying it as a very easy and instant procedure. Practice has proven otherwise; it exposed how little is known about the early development of mammals and epigenetic reprogramming. Unfortunately, somatic cell nuclear transfer success rate in mammals has not changed much since its very beginning. It is not uncommon that hundreds of oocytes need to be reconstructed to obtain a single live birth. In this review we provide a brief summary of the progress and problems of the field; beginning with selection of the donor cells and their susceptibility to different methods of epigenetic reprogramming; methods of the later gene activation, placental abnormalities, and their possible causes; to health issues that such offspring is prone to.
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The many problems of somatic cell nuclear transfer in reproductive
cloning of mammals
Katarzyna Malin
, Olga Witkowska-Piłaszewicz
, Krzysztof Papis
Department of Large Animals Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787,
Warsaw, Poland
Center for Translational Medicine, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787, Warsaw, Poland
Fertility Clinic nOvum, Warsaw, Poland
article info
Article history:
Received 27 March 2022
Received in revised form
20 June 2022
Accepted 20 June 2022
Available online 30 June 2022
In 1996, when Dolly the sheep was born, a new, utopian era was expected to begin. Science ction and
popular culture instantly threatened the public with shortly upcoming human clones, portraying it as a
very easy and instant procedure. Practice has proven otherwise; it exposed how little is known about the
early development of mammals and epigenetic reprogramming. Unfortunately, somatic cell nuclear
transfer success rate in mammals has not changed much since its very beginning. It is not uncommon
that hundreds of oocytes need to be reconstructed to obtain a single live birth. In this review we provide
a brief summary of the progress and problems of the eld; beginning with selection of the donor cells
and their susceptibility to different methods of epigenetic reprogramming; methods of the later gene
activation, placental abnormalities, and their possible causes; to health issues that such offspring is prone
©2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (
1. Introduction
Even though cloning became a matter of public discussion only
in the late twentieth century, the rst attempts were undertaken
ca. 1892; when Driesch separated blastomeres of a sea urchin
embryo and demonstrated that two separate organisms were
formed [1]. After 60 years Robert Briggs and Thomas J. King per-
formed the rst nucleus transfer, from blastula cells into enucleated
frog eggs [2], but cloning mammals hadn't been made possible for
next 45 years. In 1996, over a hundred years of research resulted in
the rst successful attempt to clone a mammal from an adult so-
matic cell [3]. Not only the science world saw it as a breakthrough;
popular media sought to portray the apparently and shortly up-
coming human cloning as the biggest threat to society, resulting in
titles such as Greetings from Frankenstein: Is the Cloned Human
on the Way?and continued to portray scientists as the insane [4].
In reality, reproductive cloning is performed by a transfer of a so-
matic cell nucleus into an enucleated oocyte and results in a single
cell, analogous to a fertilized oocyte, usually referred to as a
reconstructed oocyte. Despite the fears of uncontrolled and easy
cloning, practice shows that quarter a century later little is under-
stood in the matter and many moreyears of innovative research are
required for cloning to be as successful as in vitro fertilization (IVF)
or intracytoplasmic sperm injection (ICSI).
This year Dolly the sheep would have been 26 years old. While
many more species were reported to be cloned, overall success rate
from the creation to a viable and healthy offspring remains at a
similar, low level. In 2002, Y. Tsunoda and Y. Kato have published a
commentary, summarizing the live birth ratio as low as 0.5%e18%
in sheep, cattle, pigs and mice [5]. A review from 2007 estimated it
as low as 1e5% [6]. A nation-wide survey in Japan, that covered 9
years of Somatic Cell Nuclear Transfer (SCNT) efforts (1998e2007)
showed no improvement in development and survival until 6
months of cloned calves (on average 4.3% success rate, with 7.5% in
1998 and 2.8% in 2007) [7]. Similar statement was made as of 2020
[8]. Nonetheless, today reproductive cloning serves as a tool of
creation of champion polo horses, farm animals of exceptional
genetic makeup and as a way of bringing back beloved pet
Dolly was the only viable infant of 277 couplets created with
mammary epithelium cells [3]. In 1998, the Honolulu technique
was developed [9], resulting in the rst successful murine cloning.
*Corresponding author.
E-mail address: (O. Witkowska-
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Theriogenology 189 (2022) 246e254
There, in mice, the cumulus cells showed to be the most suitable
nuclei donors and the only ones able to produce offspring. Out of 4
experiment series, the highest birth rate was reported to reach
2.8%. The perhaps unexpected difculties caused a 5 year gap be-
tween the rst mouse and rat cloned [10]. In rats, specically, oo-
cytes activate spontaneously within an hour after being removed
from the oviducts. The original protocol, allowing for the rst
successful rat nuclear transfer followed by live births, involved
collecting the oocytes in the presence a proteasomal inhibitor
(MG132). It reversibly inhibited the oocyte from entering anaphase
II, thus preventing precocious activation. Starting with 129 recon-
structed and transferred embryos, three pups were delivered alive
and two of them survived (which marks 1,5%) and later also pro-
duced offspring [11]. It is highly desired to create rat clones pop-
ulations, as they make a useful model for medical research. As new
lines of inbred animals are created and other become excinct, to
achieve a 98.6% homozygous, isogenic population, over twenty
consecutive generations of brother x sister or parent x offspring
crossing must occur [12,13]. This process could perhaps be replaced
by somatic cell-cloned animals.
The rst cloned pet animal, CC, simultaneously the rst ever
cloned cat, was particularly famous for a different coat color than
the nuclear donor, as it is inuenced by developmental factors,
other than genetic [14]. She was also the rst clone to reproduct,
with three out of four kittens viable. The offspring and the mother
herself were reported to live in good health, and CC died in 2020 at
18 years old [15]. Many difculties had to be overcome to clone the
rst dog as well, and the specimen was born in 2005.1095 embryos
were transferred to 123 bitches and three pregnancies were
conrmed, with Snuppybeing the only viable offspring [16].
Later, in 2011, Jung et al. reported their effort to clone pekinese
dogs, where the reconstructed oocytes were activated using 4
different techniques (needle and chamber fusion methods, both
divided in groups using low and high voltage). 180 embryos were
transferred, and out of 3 fetuses only one pup was born alive (0.5%)
[17]. Despite inaccesibility to canine oocytes, relatively rare ovula-
tion and very low in vitro maturation efcacy of canine oocytes [18],
a resounding success was achieved and canine alongside feline
cloning is commercially available as of 2022 at a cost of ca. $50 000
and $35 000, respectively [19]. Recently an attempt to clone Ma-
caque Monkeys was reported [20]. With an optimized protocol, out
of 301 reconstructed embryos, four live births were obtained and
only two of them survived. This makes 1,3% birth rate, which is also
rather similar to the results from 20 years prior.
The rst ever cloned calves were obtained from only ten embryo
transfers, and of eight live births four survived [21]. The authors
speculated that no abnormalities were observed post-mortem, and
the demise was due to environmental causes, including pneumonia
following a heatstroke, drawing in superuous amniotic uid, and
one as a consequence of dystocia. Despite high success ratio of the
initial study, a 2001 report on cloning efciency, which included 13
citations of adult SCNT in cattle, showed that most of them didn't
achieve 2% live births compared to the number of reconstructed
embryos [22]. In 2007, 49.1% of 160 laboratories worldwide were
reported to clone cattle [23]. As this practice became widely
available among breeders and due to the consumers' concerns,
Food and Drug Administration (FDA) offers Consumer Health In-
formation. It addresses, among others, safety of consumption of
meat derived from cloned specimens and the fact that the cloned
animals are meant for breeding, and not direct consumption, as the
cost of obtaining such animals is also very high [24]. SCNT remains
the main method, when it comes to obtaining gene edited livestock
Prometea, the rst equine clone, was born in 2003. She was the
only viable foal obtained from 841 reconstructed zygotes [26]. In
2008, a review of SCNT progress in horses was released [27]. Au-
thors cited ve studies (all of them published from 20 03 to 2007) in
which a total of 3577 equine oocyte reconstructions were per-
formed and only 15 foals were born. Highest success rate was
achieved when donor cells were treated with roscovitine, which is
thought to synchronize the treated donor cells in G0/G1. Several
protocols of activation were additionally applied in these groups,
including ionomycin and/or sperm extract [28]. Commercial equine
cloning is also performed (ex. Viagen, Sinogene), but international
studbooks registering racing thoroughbreds refuse to recognize
clones [29]. Argentine Polo Pony clones are accepted in competi-
tions. It is claimed that Crestview, a commercial laboratory that
offers such service, cloned over 200 horses before 2016 [30] with a
30e40% pregnancy rate and 10% of the pregnancies end up with a
live birth rate [31].
Despite extreme difculties, SCNT is often seen as a hope to
restore extinct species or help preserve the endangered. In popular
media, Mammoth de-extinctionis brought up every few years as
some small progress is made in the matter [32]. Pyrean Ibex was
successfully cloned, using tissue from the last known specimen, but
the infant died shortly after birth due to left lung atelectasis. An
additional lobe was also found occupying majority of its thoracic
cavity, with no connection to the tracheobronchial tree [33].
Similarly, the rst cloned gaur died within the rst 48 h after birth
[34]. Other endangered species, including Grey Wolves [35], Black
Footed Ferret [36], Przewalski's Horse [37], were successfully
cloned. To the authors' best knowledge, as of 2022, SCNT is not
considered a major strategy in preserving any endangered species.
In Table 1, we summarized selected publications, focused on
species and corresponding success rate which illustrate two and a
half decades of advances in the eld. They are extremely valuable,
especially in the cases where the species are endangered by
2. Protocol issues
The procedure of cloning by SCNT begins with the nucleus
donor cell and the reciepient ooplast. A brief summary of the
process is illustrated in Fig. 1. While the process can be presented
generally, it needs to be recognized that in practice, there is high
species-specicity involved.
It is common for the rather older literature to suggest that
achieving G0 phase is necessary for the nucleus donor cell before it
is fused together with the ooplast [38], which can be obtained
through serum starvation. More modern evidence shows that non-
starved G1 cells serve the same purpose with success [39,40] and in
some cases, are equally successful to G0 cells [41]. From this
perspective cumulus cells might provide a great facillitation, as 80%
of them prove to be arrested in G0/G1 stage naturally and are very
suitable as nucleus donors due to the fact that no in vitro culture is
required [42,43]. On the other side of the fusion, to obtain the
ooplast, the oocyte's meiotic spindle complex is removed, requiring
a proper visualization technique. For example, bovine oocytes can
be treated with UV and uorochrome bisbenzimide (Hoechst
33342) staining, to which they are relatively resistant [44]. Primate
embryos only show comparable blastocyst formation rate (15e16%)
when Oosight imaging system is introduced, which does not
involve the staining. A speculation was raised by the authors, that
Hoechst 33342 might activate primate oocytes prematurely or
cause degradation of maturation factors, which could be later
linked to epigenetic reprogramming failure [45]. Later, the positive
impact of replacing staining with Oosight imaging system in bovine
embryos was also demonstrated [46]. In mice specically, the
spindle complex can be made visible using Nomarski or Hoffman
optics, also without staining, in 37
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
There is an ongoing debate, whether oocytes stripped of Zona
Pellucida are better candidates for this process, as SCNT using Zona
Pellucida-Free (ZF) oocytes tends to be simpler and quicker [48]. It
seems to have no affect on bovine [49,50], equine [26,51] and
murine embryo development [52]. However, it does not apply to
any species given - in cats, removing zona pellucida later leads to
complete impairment and failure of such embryos' implantation
[53]. ZF embryos require individual, longer lasting culture, i.e., until
the blastocyst develops. Additionally, treating the cells with
phytohemagglutinin further increases the chances of both cells'
fusion [54]. Zona-enclosed embryos, which are harder to obtain,
can be transferred at cleavage [54]. Regarding the difculties with
ZF oocytes in cats, specic gene expression abnormalities were
identied during the in vitro culture of such: SOX2 and NANOG -
considered as markers of pluripotency expression - were under-
expressed and BAX, apoptosis marker, proved overexpressed [53].
To authorsbest knowledge, there are no successful cases reported
of ZF feline cloning.
The metaphase II stage arrested, and enucleated oocytes
become the recipients of the donor nucleus, whether it is injected
or the cells are fused [55]. Until their activation, the development is
managed by factors which were already present in the recipient
oocyte, such as Maturation/Meiosis/Mitosis-Promoting Factor
(MPF) [56]. Physiologically, sperm cells introduce Phospholipase C
Zeta 1 (PLCZ1, absent in somatic cells), which, through calcium
oscillation and MPF breakdown, induces the meiotic arrest release
and further development [57]. Alike fertilized oocytes, the recon-
structed oocytes need to be activated to enter the division cycle.
Table 1
Summary of the species and corresponding success rate.
Year Species Results Other comments
1997 [3] Sheep Dolly the sheep was born; the only viable offspring of 277
The rst mammal cloned from adult somatic cells
1998 [9] Mouse Four experiments, highest birth rate 2.8% First mice cloned
2003 [10] Rat 3 pups born of 129 reconstructed embryos First rats cloned, later reproduced naturally
2002 [14] Cat One viable offspring of 82 transferred embryos, total 188
reconstructed oocytes
The rst cat cloned, the rst pet cloned, later produced healthy offspring.
She died at the age of 18 due to kidney failure [130]
2005 [16] Dog Three pregnancies obtained from 1095 embryos
transferred; one viable offspring
Snuppy the dog was later cloned as well [131]
2011 [17] Dog One viable offspring of 180 transferred embryos Four different activation techniques were tested in this experiment
2007 [35] Wolf 251 reconstructed embryos, 2 live births Endangered species; Grey Wolf somatic cells nuclei were transferred to
canine oocytes
2003 [26] Horse Only one viable foal obtained from 841 embryos First cloned horse
2016 [109] Horse 16 blastocysts resulted in 11 pregnancies, 3 foals delivered
All of the placentas showed abnormalities
2020 [127] Rabbit 61 blastocysts formed from 165 reconstructed oocytes This result was achieved using electrofusion and melatonin used as a
protective agent against electrofusion damage
2020 [36] Black footed
1 live birth Donor cells cryopreserved over 30 years prior to the procedure.
2021 [128] Dromedary
1033 reconstructed oocytes, 28 births, 19 calves survived No signicant differences in effectiveness regarding the breed of the oocyte
2020 [37] Przewalski's
One foal born to a domestic horse dame Cells cryopreserved in 1980 were used in this procedure
2019 [100] Crab eating
5 live births out of 325 embryos transferred Involved (CRISPR)/Cas9 editing
Fig. 1. A general illustration of the SCNT protocol.
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
This process must be recreated articially and so far multiple
activation methods have been investigated.
The protocol which resulted in Dolly's birth consisted of elec-
trical pulses between 34th and 36th hour after the ovulation was
induced in donor ewes [3]. In the aforementioned Honolulu tech-
nique, best results were achieved when the oocytes were activated
one to 3 h after the nucleus transfer, in calcium-free, 10 mM Sr
and 5
cytochalasin B environment [9]. Other reports
describe PLCZ cRNA injection, which results in repetitive
fertilization-like Ca
rises in bovine reconstructed oocytes [58].
This method was tested alongside with two more common pro-
tocols involving ionomycin/6-dimethylaminopurine (DMAP) and
ionomycin/cycloheximide (CHX). In this study, PLCZ showed lower,
but comparable efciency (27.7% embryos developed to blastocyst
stage vs. 35.9% for ionomycin/CHX). Another study describes a
comparison of three activation methods in porcine embryos:
electrical fusion/activation, electrical fusion/activation combined
with MG132, and electrical fusion in low Ca
followed by
chemical activation with thimerosal/dithiothreitol. The second
treatment was the most effective, but only 2 piglets (1.9% of em-
bryos transferred) were delivered [59]. It was also proposed to
activate the reconstructed oocyte by reducing zinc ions concen-
tration within the cell. As a result, even in the absence of cellular
changes, the oocyte can be activated, when Zn
is depleted
with heavy metal chelators in the intercellular space [60].
In 2019 another study was published, which focused on creating
a faster but equally reliable porcine protocol. Four experiments took
place and were compared to a control group, which was treated
with the routine protocol for the laboratory (ionomycin exposure
for 5 min followed by SrCl
and CHX for 4 h, total 245min). It was
concluded that similar percentage of embryos which developed to
the blastocyst stage was achieved with all methods, including a
20 min procedure, where oocytes were exposed to ionomycin for
5 min plus 15 min of TPEN (N,N,N0,N0-Tetrakis(2-pyridylmethyl)
ethylenediamine) treatment [61]. Unfortunately, time-saving pro-
cedures might not be as benecial after all, as a 2019 meta-analysis
reveals that delaying the activation by over an hour signicantly
increases the blastocyst formation rate [62].
3. Epigenetics
It has been clear for decades that the biggest challenge
regarding the cloned embryo development lays in its epigenetic
reprogramming; physically, among many, resulting in hardships
regarding the implantation, placenta development and function,
but also abnormal offspring syndrome [63], later obesity, immu-
nodeciency, respiratory defects and early death [64]. It has been
concluded that this epigenetics issue is vastly responsible for the
low birth rate [65,66]. It is critical for the cell nucleus to return to
totipotency through undifferentiation. If incomplete before
genome activation, the failed patterns will be copied and expressed,
resulting in fetal abnormalities and death [67,68]. Dysregulations at
the time of genome activation have been studied and multiple
aberrations have been proven. As many as 259 differently expressed
mRNAs have been reported in a study only at the one cell stage of an
SCNT embryo, compared to in vivo fertilized embryos, making ca.
1.6% of all detected transcripts (137 of higher, 122 of lower
expression) [69].
As mentioned, no epigenetic manipulations were applied in the
murine protocol [9]. In case of Dolly, the cell cultures were serum-
starved only with a suggestion, that such quiescence could possibly
help the reprogramming [3]. Both cases show that the oocyte itself
has the ability to reprogram the transferred nucleus. To increase the
birth rate pharmaceutical intervention can be applied. Among
many, DNA methylation, histone acetylation and methylation were
studied as potential molecular targets with a variety of results.
Trichostatin A (TSA) is a drug of Histone Deacetylase Inhibitor
(HDACi) class [70]. Its action, through interfering with enzymatic
removal of acetyl groups from histones, assures the DNA accessi-
bility for the transcription factors. In case of somatic cell nucleus
remodeling, the addition of TSA is expected to support unfolding of
the nucleosome so it can resemble the totipotent pattern. In 2006,
two groups, independently, have established the most appropriate
protocol for murine SCNT using TSA and vefold increase in the
embryo's development was reported [71,72]. Another report
delivered data of enhanced blastocyst development rate in monkey
(4%e18%) [73]. Embryos reconstructed using adult bone marrow
Mesenchymal Stem Cells (MSC)and treated with TSA prove the
success rate to grow exponentially, with 65,2% of electrofused
embryos reaching the blastocyst stage, of higher cytological and
molecular quality compared to 45,5% in those untreated [74 ].
Nonetheless, questions were raised about TSA's toxicity and tera-
togenicity [75]. In a recent meta-analysis of its effectiveness on pig
SCNT blastocysts yield, an important conclusion was drawn, that
TSA improves the embryo development when the nuclear donor
cell is of fetal and not adult source. Furthermore, even though the
embryonic development is usually increased, births are reduced
[76]. In rabbits, a study was performed where out of six recipients
two became pregnant with reconstructed embryos treated with
TSA [77]. Out of eleven pups, seven were born alive but all died
within an hour to 19 h. The authors recommend further investi-
gation on long-term effects of such treatment, but don't associate
the mortality with it as early deaths are common in cloned animals.
Another substance of the same class, Scriptaid, has been shown to
provide similar therapeutic effects [78] with less toxicity [79] but a
different study showed improved blastocyst formation in bovine
embryos only when treated with TSA and not Scriptaid [80]. A 2022
meta-analysis on Scriptaid's potency to increase the number of
blastocyst cells or the cleavage rate in porcine embryos derived
from SCNT concluded that only the former might be true [81].
Other epigenetic treatment candidates researched include
Chetomin, a fungal secondary metabolite (which supposably in-
hibits the trimethylation on histone 3 lysine 9 (H3K9 me3)) and
caffeine (as a protein phosphatase inhibitor) [82]. In the cited article
authors did not observe any improvement in bovine and equine
embryo development compared to TSA, even after a 48 h exposure
to Chetomin. Caffeine, on the other hand, was shown to improve
the developmental capability of porcine embryos [83] and primate
embryos [84]. Another H3K9 methyltransferase inhibitor, Chaeto-
cin, was studied but opposite to expectations, it impaired the ovine
embryo development [85]. More recently, a different, promising
approach on epigenetics was proposed: Mutliple Barriers Removal
[86]. The following was proposed: Histone 3 lysine 9 trimethyla-
tion, the rst barrier, can be removed by injecting a demethylase
(Kdm4d) mRNA into reconstructed embryos 5 h post-activation.
Similarly can be done with Kdm5b mRNA injection into the
enucleated oocyte. Combining both increased the rate of murine
cloned embryosdevelopment into live animals to 11% [87]. Also
knock-out of H3K9me3 transferases in the donor cells prior to SCNT
brought improvement of almost 50% in blastocyst formation
compared to 6,7% in control group [88]. Authors point out reme-
thylation to be another barrier, which could be overcome by
injecting siRNAs of methyltransferases Dnmt3a and Dnmt3b into
the enucleated oocytes. Finally, the same authors reported that
92.3% of enucleated oocytes that underwent Kdm4bþ5b mRNA and
siDnmt3aþ3b co-injection reached the blastocyst stage. So far no
data is available whether any viable offspring was ever obtained
using this method. Unfortunately, promising embryonic develop-
ment during the in vitro culture might have nothing to do with
survival until birth and later viability of such newborn. In
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
epigenetics, it is perfectly illustrated when donor cells of different
origin are used: equine adult skin broblasts show similar blasto-
cyst development rate, and even birth rates to equine MSC, but foals
created with MSC are not burdened with malformations or diseases
and present higher viability [89]. This might be due to the fact that
MSC show lower activity of Histone Deacetylases and DNA meth-
yltransferases (DNMTs) [74], which is possibly the reason for the
susceptibility to epigenetic reprogramming. Using MSC as the nu-
cleus donors also seems to improve the success rate of the pro-
cedure in equines ein a study where MSC (along with adult
broblasts) were compared as donor cells against a control group of
Articial Insemination (AI) embryos. A total of 594 MSC embryo-
transfers resulted in 37 pregnancies and 20 viable foals (3,4% suc-
cess rate, compared to 1,9% for adult broblasts and 71,6% for
embryo transfer) [89]. Good developmental perspectives have been
shown in porcine [90e92], caprine [93] and bovine embryos [94]
cloned with MSC.
4. Abnormal placental development
Although the placenta developed de novo many times
throughout the evolutionary history [95], it is usually associated
with Placentalia, a subdivision of the class Mammalia. In the live
bearing, the placenta allows for the embryo to implant in the
uterine wall; this process, if imperfect, may result in multiple pa-
thologies. Generally, they are inevitable in SCNT pregnancies. Due
to this reason, the majority of them are lost during the rst
trimester [96] or peri-implantation [97]. In bovine embryos, a
reduced number of placentomes, hydroallantois and general
enlargement are observed [98,99]. In 2006 genetic aberrations in
the developing placenta have been proven. Authors conrmed that
certain genes responsible for the trophoblast proliferation, differ-
entiation and function were expressed differently to the control
group (embryos derived from AI and IVF, leaning towards high
proliferation genes expression and low or absent function-
associated ones both pre- and post-implantation. At day 17 the
SCNT-derived trophoblast showed no signs of interferon-tau pro-
duction, which is the pregnancy recognition signal in cattle [100].
Another study identied 123 differentially expressed circRNAs,
which were found related to 60 host genes and 32 miRNAs [101].
Especially the miRNA abnormality is to be linked to the problematic
epigenetics of SCNT. In the cited study, transcriptomes of the mRNA,
lncRNA and miRNA of placental cotyledon tissue at day 180 after
gestation were sequenced and 362 mRNAs, 1272 lncRNAs and nine
miRNAs were found to differ in expression [102]. Thus, the prob-
lematic placental development is most likely emergent and not
Placentas and fetal membranes of SCNT animals have also been
examined by ultrasound imaging and histologically which revealed
even more abnormalities [103]. Amniotic membranes, pla-
centomes, umbilical cords, and fetal uid were studied by ultra-
sound and compared to an AI control group. It disclosed focal
edema, larger placentomes and increased umbilical cord diameter
with hyper-echodensity areas and spikes around it. Histological
ndings included degenerated inammatory cells, chorioallantoic
membrane edema, and decreased epithelial thickness. Clinical ob-
servations prior to the study fall into place with it, as placentome
hypertrophy, enlarged umbilical vessels and placental edema were
reported [104,105].
In mice, placental hyperplasia and expanded spongiotropho-
blast layer develop [106] with poor proliferation rates of the
trophoblast cells followed by rapid growth in later stages [107].
Recently, causes of this enlargement were studied [108]. In natural
conception in mice, genes Sfmbt2, Gab1, and Slc38a4 are paternally
expressed, but in SCNT placentas, they are expressed biallelically. At
rst, a maternal knockout of previously recognized enlargement-
related genes (Sfmbt2, Gab1, Slc38a4) was performed with no
improvement in placental quality but correcting the expression of
clustered miRNAs within the Sfmbt2 gene the physiological
placental phenotype was restored. The pups grew into normal,
fertile adults. Although after miRNA knockout the birth rate
increased more than twice (6.7% vs 3% in the wild SCNT clones) the
numbers were considered statistically insignicant. Therefore,
even though the abnormal placental development can at least be
partially prevented, more underlying issues are probably to be
In equines the placenta faces developmental issues as well. A
detailed study of equine SCNT pregnancies was published in 2016
[109]. Nuclei obtained from broblasts of a 29-years old mare were
transferred. Sixteen blastocysts underwent an embryo transfer,
resulting in eleven pregnancies, during which the placentas were
monitored for abnormalities. Three foals were delivered viable.
Pathologies were found in all assessed placentas; even in the three
successful pregnancies. In these, placental edema, engorged allan-
toic vessels and a large umbilical vessel were found. Two mares
were diagnosed with bacterial placentitis and treated with
trimethoprim sulfamethoxazole, pentoxifylline and unixin
meglumine. In the ve mares that experienced abortion the pla-
centas were found heavier than normal, up to 35% of the fetal
weight. All of them showed some form of placentitis. Villous hy-
poplasia, atrophy or necrosis, vasculopathy of allantoic and um-
bilical vessels, hemorrhages and placental mineralization were
reported as well. Additionally, four of ve umbilical cords were
longer than regular and edema, hemorrhage and mural thickening
of the umbilical vessels were observed. There were four cases of
large allantoic vesicles. Severe placental separation was diagnosed
in some as well. Two more mares were found to have had bacterial
placentitis only after the abortion, despite no ultrasonographic
ndings. The viable foals experienced hypoxic-ischemic encepha-
lopathy, pneumonia, omphalophlebitis and omphaloarteritis. Two
foals had umbilical hernias blood clots in the urinary bladders.
Incomplete calcication of carpal bones and multiple rib fractures
were found in one foal. Two foals were discharged on day 12, the
third one suffered hemothorax due to a fractured rib causing
puncture of the pericardium and was euthanized. These ndings
are mostly consistent with previous research [110 ,111], but this
particular study was the rst one to report placentitis in SCNT
5. Other ndings
Even though the majority of cloned animals live in good health,
it is estimated that nearly one in three dies within the rst 6
months of life [112 ]. Large/Abnormal Offspring Syndrome (LOS),
respiratory failure, abnormal kidney development, cardiovascular
and liver pathologies are often reported [22,112,113]. LOS is widely
associated with cloned and IVF offspring. By denition, it results in
a phenotype consisting of excessive overall fetus size, muscular
deformities, abnormal and asynchronous organ growth combined
with placental dysfunctions. Its occurrence is unpredictable [114],
but so far has not been observed in horses [27], when overgrowth is
expected, parturition induction can be performed a week before
the due date [115 ]. Abnormal fetal development is linked to obesity
later in life in animals [116] and adult murine clones struggle with
increased body mass [117].
Il-Hwa Hong et al. [118 ] reported necropsy ndings in 12 still-
born/deceased SCNT canine neonates. Signicant increase in mus-
cle mass and macroglossia were commonly observed. The tongue
size caused airway obstruction in many cases. Moreover, anterior
abdominal wall defects, edema and atelectasis of the lung tissue,
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
increased heart and liver size were present in almost all of the dogs
and one case of failed cerebrum medulla development was found.
Interestingly, this particular neonate presented normal muscle
mass and tongue size. These abnormalities raised questions on
myostatin expression in such specimens, as myostatin-decient
animals share such phenotype. The team found reduced expres-
sion in myostatin in their tongues and muscle and provided some
interesting discussion. They explain that myostatin is one of the
family members, which is regulated by IGFs during cell cycle
and its differentiation. Another study showed variable, abnormally
high IGF2R, IGF1R mRNA expression in adult SCNT produced goats
[119], otherwise healthy. IGF2 mRNA expression also varies in
bovine clones [120].
Aging in animals cloned from adult somatic cells is researched
due to justied concerns of the cloned offspring to be at the bio-
logical age of the nucleus donor at birth. Among the most con-
cerning are probably the epigenetic dysregulations, problematic in
these specimen as discussed above. Telomere length also rose
questions, as they were expected to be shorter, proportionally to
the age of the donor of the nucleus. Dolly's telomeres were
conrmed to be 20% shorter to the control group [121], and it seems
to be a tendency in cloned sheep, according to a 2017 review [122].
Surprisingly, it also reported telomere length in cloned cattle to be
appropriate for their age or even elongated, but overall, in other
species it was often found reduced compared to natural offspring of
the same age. Recent study in Dromedary Camels showed that
telomere lengths in clones was adequate to the control group [123].
Interestingly, TSA was proved to facilitate telomere elongation in
cloned pigs [124].
The aforementioned abnormal placental development might be
linked to cardiovascular issues in the early life, according to
Batchelder et al. [125]. The study suggests that anomalous placental
membranes, causing abnormal circulatory pressure, interfere with
the fetal heart's development. This thesis was supported by echo-
cardiographic ndings (initially decreased aortic diameters and
increased myocardial thicknesses) in seven cloned calves, which
showed that during the rst 30 ex utero life, they made adjustments
and by the end of the study the differences to the control group
resolved. Other authors also agree that although SCNT animals vary
from non-SCNT control offspring in the rst two months of life, it is
no longer the case after this period is over [112]. In another report,
seven of ten cloned piglets were similarly evaluated between 40
and 65 day of life [126]. Decrease in fraction ejection, mitrial
insufciency and decreased cardiac output was observed in some.
Authors also included necropsy ndings in ve piglets who died
during the study and in four of them, cardiovascular deviations
were observed, including an enlarged heart with dilated, thin-
walled ventricles with myocardial necrosis, myocardial degenera-
tion and excess pericardial uid and congestive heart failure. The
surviving ve piglets showed no symptoms of cardiac disease.
General examination, hormonal and hematologic assessments
have been performed in cattle cloned from somatic cells [127].
Fetuses obtained before term showed variable organ sizes and
deformities, including abnormally large kidney, autolysis of both
kidneys in a stillborn and large fatty liver. Neonates had higher
average birth weight compared to the AI and IVP control groups.
Rectal temperature of clones was found to be higher until 50 days of
age, including random 24e36 h peaks up to 41
C with no other
clinical signs observed, non-reactive to any treatment applied
(NSAIDs, wet towels and ventilation). T
plasma levels were
examined in speculation, whether it could be the reason for the
fever, but the answer was negative. RBC parameters were found to
be consistent within all groups. The neutrophil:lymphocyte ratio,
reecting cortisol levels at birth, was higher in clones. Despite this,
later ACTH stimulation tests found no signicant differences.
Regarding the growth hormone, normal level was found, as well as
in insulin and glucose responses. To the authorsbest knowledge,
these macroscopic ndings are not found to originate within any
specic developmental deviation.
6. Suggestions for further research
In a short summary, the low efciency reects how complicated
SCNT and further embryo development is. Some recommendations
and assumptions can be made with a precaution, that even though
sharing similar problems and results, probably no two studies can
be directly compared. Different methods and approaches are
researched in order to further rectify the whole procedure as such.
A faster and simplied SCNT would limit both oocyte's and donor
cells' exposure to external factors and manipulations and require
shorter training of the staff with similar success rates. Here, an
inevitable conclusion has to be stated - that more successful cloning
will require deeper understanding of oocyte's physiology, not only
as a mammal oocyte, but also the species specicity and unique-
ness. Especially the relationship between nucleus-donor cell type
and introduced pharmaceuticals need further studies; which might
be very complicated due to multiple possibilities and combinations.
Perhaps the future of SCNT lies in the Mutliple Barrier Removal
technique, as it potentially withdraws the need of using any clas-
sical pharmaceutical treatment, but on the other hand, may require
more injections per se. Yet again, blastocyst formation or cleavage
rate, often presented as determinants of a successful study, may not
have anything to do with the embryo's pregnancy survival and the
newborn's viability. While many studies are promising, their actual
value can only be veried by obtaining healthy offspring.
Aside from discussed technical difculties, ethics of cloning,
widely discussed in the popular media, cannot be forgotten. So far
no successful human reproductive cloning attempt was docu-
mented, not only due of the legal regulations, but also due to lack of
reasonable cause for such; when it comes to infertility, multiple
methods other than SCNT can be used. In animal cloning, doubts
could be raised especially in case of commercial dog cloning. World
famous Snuppy, the rst dog cloned by SCNT, out of 1000 embryos
transferred into 123 surrogates, was the only pup surviving until
adulthood [16]. To our knowledge, such businesses do not publicly
share information on the surrogatesliving conditions, especially
those, who experience abortion, nor about the dogs that do not get
adopted due to lack of predictability regarding the live births count.
Despite multiple issues, of which some we briey described,
progress continuously takes place. In the end, SCNT may not be as
easy as portrayed quarter a century ago, but each and every advance
may be a giant leap for the science of medicine.
Author contributions
Conceptualization: K.M., O.W.P., K.P.; Roles/Writing - original
draft: K.M., O.W.P. Writing - review &editing: K.M., O.W.P., K.P.
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors.
Declaration of competing interest
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... Sci. 2022, 23, 15975 2 of 17 jade1, Gab1, and smoc1 [8,10,11]. Correction of these reprogramming errors have been shown to dramatically enhance animal cloning efficiency [8,10,11]. ...
... 2022, 23, 15975 2 of 17 jade1, Gab1, and smoc1 [8,10,11]. Correction of these reprogramming errors have been shown to dramatically enhance animal cloning efficiency [8,10,11]. ...
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The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos.
... The developmental competence of embryos derived from somatic cell nuclear transfer (NT) is lower compared to those from parthenogenetic activation (PA), in vitro fertilization (IVF), or intracytoplasmic sperm injection [1,2]. NT success rate in mammals has not changed significantly since the beginning [3,4]. Many factors are involved in the low pregnancy rate [5]. ...
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The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.
... Once again, blastocyst development or cleavage rate, which are usually mentioned as success indicators in research, may not be significantly correlated with the survivability of the embryo during pregnancy. Even though there is a lot of promising research, their true value can only be gauged by how many healthy offspring they can produce [62]. One of the factors that has been connected to cloning failure is a higher rate of oocyte degradation. ...
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Cloning, commonly referred to as somatic cell nuclear transfer (SCNT), is the technique of enucleating an oocyte and injecting a somatic cell into it. This study was carried out with interspecific SCNT technology to clone the Arabian Oryx utilizing the oryx’s fibroblast cells and transfer it to the enucleated oocytes of a domestic cow. The recipient oocytes were extracted from the cows that had been butchered. Oryx somatic nuclei were introduced into cow oocytes to produce embryonic cells. The study was conducted on three groups, Oryx interspecific somatic cell nuclear transfer into enucleated oocytes of domestic cows, cow SCNT “the same bovine family species”, used as a control group, and in vitro fertilized (IVF) cows to verify all media used in this work. The rates of different embryo developmental stages varied slightly (from 1- cell to morula stage). Additionally, the oryx interspecies Somatic cell nuclear transfer blastocyst developmental rate (9.23%) was comparable to that of cow SCNT (8.33%). While the blastula stage rate of the (IVF) cow embryos exhibited a higher cleavage rate (42%) in the embryo development stage. The results of this study enhanced domestic cow oocytes’ ability to support interspecific SCNT cloned oryx, and generate a viable embryo that can advance to the blastula stage.
... successful applications have been demonstrated, including intracytoplasmic sperm injection (ICSI) [7], [8], nuclear transfer (animal clone) [7], [10], genomic testing [11], [12] and preimplantation genetic diagnosis (PGD) [13], [14], [15]. Micropipettes are the conventional tools used for cell transportation. ...
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Single-cell transportation is one of the most common cell operations. Transporting cells with micropipettes is convenient for a wide range of biomedical applications. For high-efficiency cell transportation, the cells must be aspirated into the orifice of a micropipette. However, this is very difficult to achieve, as there is relative movement between the cell and the culture medium when the fluid drives the cell in the culture medium. It is crucial to use cell dynamics rather than fluid dynamics as the control objects to improve control performance and stop the cell immediately when it approaches the micropipette. In this study, the cell dynamics were modeled using a second-order model by integrating the dynamic model between the fluid and the cell into a first-order fluid dynamic model. A backstepping controller-based extended state observer was proposed to control the cell movement inside the micropipette. Experiments demonstrated that the proposed controller could aspirate cells into the orifice of the micropipette with high accuracy and no overshoot. Furthermore, the proposed controller was applied to automated somatic cell nuclear transfer, and it significantly boosted operational efficiency. Note to Practitioners —The need to apply advanced automation methods to transfer cells in life sciences has increased at a steady pace. The key feature of such systems is the ability to select and transfer cells at a predetermined position in space and time for biological applications. We propose a cell positioning control method based on vision-guided robotics that can directly aspirate cells to specified positions near the orifice of a micropipette. In somatic cell nuclear transfer, the proposed method of transferring somatic cells into oocytes occurs at a faster pace than manual operation. This provides essential functionality for single-cell transfer and is an appropriate technology for practitioners with this functional requirement.
Low cloning efficiency limits the wide application of somatic cell nuclear transfer technology. Apoptosis and incomplete DNA methylation reprogramming of pluripotency genes are considered as the main causes for low cloning efficiency. Astaxanthin (AST), a powerfully antioxidative and antiapoptotic carotenoid, is recently shown to improve the development of early embryos, however, the potential role of AST during the development of cloned embryos remains unclear. This study displayed that treating cloned embryos with AST significantly increased the blastocyst rate and total blastocyst cell number in a concentration dependent manner, and also alleviated the damage of H2O2 to the development of cloned embryos. In addition, compared with the control group, AST significantly reduced the apoptotic cell number and rate in cloned blastocysts, and the significantly upregulated expression of anti-apoptotic gene Bcl2l1 and antioxidative genes (Sod1 and Gpx4) and downregulated transcription of pro-apoptotic genes (Bax, P53 and Caspase3) were observed in the AST group. Moreover, AST treatment facilitated DNA demethylation of pluripotency genes (Pou5f1, Nanog and Sox2), in accompany with the improved transcription levels of DNA methylation reprogramming genes (Tet1, Tet3, Dnmt1, Dnmt3a and Dnmt3b) in cloned embryos, and then, the significantly upregulated expression levels of embryo development related genes including Pou5f1, Nanog, Sox2 and Cdx2 were observed in comparison with the control group. In conclusion, these results revealed that astaxanthin enhanced the developmental potential of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes, and provided a promising approach to improve cloning efficiency.
The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.
Purpose: to conduct a comparative analysis of the effect of commercial media BO-IVC and СR1aa at the stage of the activation and subsequent culture of artificially activated oocytes on the formation and quality of parthenogenetic bovine embryos. Materials and methods. 3 groups of disemeters of 50 goals in each were formed. In the first experimental group, the disemeted was in a meticulous manner with a ram-industrialist (artificial kriproporchid), in the second experimental-with a penEexctomed ram-industrialist. In the third (control) group, a producer ram was used. In the first experimental group of a ram-industrialist (artificial kriproporchid) with attached taps were released into a group of sheep twice a day for 1.5-2 hours. In the second experimental group of a penEctomed ram, it was placed in the corral to the disemetery in the morning for 3 hours. In the third group, the lamb producer was constantly with the disemets for two weeks, then he was changed on a new ram i.e. Used the methodology used in the farm. During the experiment, they observed the behavior of animals of all groups. In the experimental groups, after the detection of disemeters in the hunt, their natural insemination of the manufacturer was carried out. Based on the results of the subsequent oster, the effectiveness of the reproduction of sheep was evaluated. Results. The cleavage rate did not differ between the experimental groups, varying from 73,0 to 76,5%. Also, there was not found a significant effect of the conditions for post-activation culture of oocytes on their development before late morula and late blastocyst stage, which was for the CR1aa/CR1aa, CR1aa/BO-IVC and BOIVC/ BO-IVC groups 28,9±1,7, 40,4±7,5 and 36,0±6.4%, respectively. Meanwhile, we found out the effect of tested culture conditions on the ability of parthenogenetic embryos to overcome the 8-16 cell block and their quality on the late stages of embryo development. The rate of embryos with less than 16 nuclei was the highest in the CR1aa/CR1aa group (56,8±2,1 %). The replacement of CR1aa medium to BO-IVC medium (BO-IVC/BO-IVC group) significantly reduced this level (p<0,05). The positive effect was enhanced when CR1aa medium was used at the stage of culture in the presence of 6-DMAP and cycloheximide, and subsequent embryo development was in BO-IVC medium (CR1aa/BO-IVC group) (p<0.001). Furthermore, when we used the mixed variant of culture, the total cell number in parthenogenetic morula and blastocyst stage embryos increased (p<0.05). Conclusion. Thus, the BO-IVC medium at the stages of post-activation and subsequent development of artificially activated bovine oocytes is comparable to the CR1aa medium in terms of the efficiency of obtaining parthenogenetic embryos at the blastocyst stage. Nevertheless, its replacement at the post-activation stage with CR1aa medium makes it possible to improve the quality of parthenogenetic embryos.
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Cloned animals generated by the somatic cell nuclear transfer (SCNT) approach are valuable for the farm animal industry and biomedical science. Nevertheless, the extremely low developmental efficiency of cloned embryos hinders the application of SCNT. Low developmental competence is related to the higher apoptosis level in cloned embryos than in fertilization-derived counterparts. Interleukin 17D (IL17D) expression is up-regulated during early mouse embryo development and is required for normal development of mouse embryos by inhibiting apoptosis. This study aimed to investigate whether IL17D plays roles in regulating pig SCNT embryo development. Supplementation of IL17D to culture medium improved the developmental competence and decreased the cell apoptosis level in cloned porcine embryos. The transcriptome data indicated that IL17D activated apoptosis-associated pathways and promoted global gene expression at embryonic genome activation (EGA) stage in treated pig SCNT embryos. Treating pig SCNT embryos with IL17D up-regulated expression of GADD45B, which is functional in inhibiting apoptosis and promoting EGA. Overexpression of GADD45B enhanced the developmental efficiency of cloned pig embryos. These results suggested that IL17D treatment enhanced the developmental ability of cloned pig embryos by suppressing apoptosis and promoting EGA, which was related to the up-regulation of GADD45B expression. This study demonstrated the roles of IL17D in early development of porcine SCNT embryos and provided a new approach to improve the developmental efficiency of cloned porcine embryos.
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Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.
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Somatic cell nuclear transfer (SCNT) enables terminally differentiated somatic cells to gain totipotency. Many species are successfully cloned up to date, including nonhuman primate. With this technology, not only the protection of endangered animals but also human therapeutics is going to be a reality. However, the low efficiency of the SCNT-mediated reprogramming and the defects of extraembryonic tissues as well as abnormalities of cloned individuals limit the application of reproductive cloning on animals. Also, due to the scarcity of human oocytes, low efficiency of blastocyst development and embryonic stem cell line derivation from nuclear transfer embryo (ntESCs), it is far away from the application of this technology on human therapeutics to date. In recent years, multiple epigenetic barriers are reported, which gives us clues to improve reprogramming efficiency. Here, we reviewed the reprogramming process and reprogramming defects of several important epigenetic marks and highlighted epigenetic barriers that may lead to the aberrant reprogramming. Finally, we give our insights into improving the efficiency and quality of SCNT-mediated reprogramming.
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Accelerated development of novel CRISPR/Cas9-based genome editing techniques provides a feasible approach to introduce a variety of precise modifications in the mammalian genome, including introduction of multiple edits simultaneously, efficient insertion of long DNA sequences into specific targeted loci as well as performing nucleotide transitions and transversions. Thus, the CRISPR/Cas9 tool has become the method of choice for introducing genome alterations in livestock species. The list of new CRISPR/Cas9-based genome editing tools is constantly expanding. Here, we discuss the methods developed to improve efficiency and specificity of gene editing tools as well as approaches that can be employed for gene regulation, base editing, and epigenetic modifications. Additionally, advantages and disadvantages of two primary methods used for the production of gene-edited farm animals: somatic cell nuclear transfer (SCNT or cloning) and zygote manipulations will be discussed. Furthermore, we will review agricultural and biomedical applications of gene editing technology.
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Somatic cell nuclear transfer (SCNT) in mammals is an inefficient process that is frequently associated with abnormal phenotypes, especially in placentas. Recent studies demonstrated that mouse SCNT placentas completely lack histone methylation (H3K27me3)-dependent imprinting, but how it affects placental development remains unclear. Here, we provide evidence that the loss of H3K27me3 imprinting is responsible for abnormal placental enlargement and low birth rates following SCNT, through upregulation of imprinted miRNAs. When we restore the normal paternal expression of H3K27me3-dependent imprinted genes (Sfmbt2, Gab1, and Slc38a4) in SCNT placentas by maternal knockout, the placentas remain enlarged. Intriguingly, correcting the expression of clustered miRNAs within the Sfmbt2 gene ameliorates the placental phenotype. Importantly, their target genes, which are confirmed to cause SCNT-like placental histology, recover their expression level. The birth rates increase about twofold. Thus, we identify loss of H3K27me3 imprinting as an epigenetic error that compromises embryo development following SCNT. Somatic cell nuclear transfer (SCNT) frequently results in abnormal placenta development in cloned mice. Here the authors show that loss of histone methylation (H3K27me3) imprinting in clustered Sfmbt2 miRNAs contributes to SCNT placenta defect.
Context: Incomplete epigenetic reprogramming of histone deacetylation (HDAC) is one of the main reasons for the low efficiency of somatic cell nuclear transfer (SCNT). Scriptaid is a synthetic HDAC inhibitor (HDACi) that may improve the efficiency of porcine SCNT. Aims: This study aimed to determine whether scriptaid increases the number of blastocyst cells or the cleavage rate. Methods: We conducted a meta-analysis of the pertinent literature published over the past decade. Key results: A total of 73 relevant papers were retrieved using a diverse English keyword search, and 11 articles were used for the meta-analysis. Scriptaid was positively correlated with blastocyst rate but had no effect on cleavage rate or blastocyst cell number. A subgroup analysis of blastocyst cell number showed that the staining method was the source of the heterogeneity. Conclusions: In SCNT embryos, scriptaid treatment after activation can promote embryonic development, but there may be adverse effects on early development. Implications: HDACi research should focus on SCNT birth efficiency.
Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer (SCNT) and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+ based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.
There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.
The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 μm) than that from the ZI group (210.5 ± 78.5 μm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.