BIOLOGY OF REPRODUCTION (2012) 86(6):180, 1–6
Published online before print 4 April 2012.
DOI 10.1095/biolreprod.111.098764
Latrunculin A Can Improve the Birth Rate of Cloned Mice and Simplify the Nuclear
Transfer Protocol by Gently Inhibiting Actin Polymerization1
Yukari Terashita,3,4,5 Sayaka Wakayama,4 Kazuo Yamagata,4,6 Chong Li,4 Eimei Sato,5 and Teruhiko
Wakayama2,4
4
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Kobe, Japan
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
6
Center for Genetic Analysis of Biological Responses, Research Institute for Microbial Diseases, Osaka University,
Osaka, Japan
5
Although animal cloning is becoming more practicable, there
are many abnormalities in cloned embryos, and the success rate
of producing live animals by cloning has been low. Here, we
focused on the procedure for preventing pseudo-second polar
body extrusion from somatic cell nuclear transfer (SCNT)derived oocytes. Typically, reconstructed oocytes are treated
with cytochalasin B (CB), but here latrunculin A (LatA) was used
instead of CB to prevent pseudo-second polar body extrusion by
inhibiting actin polymerization. CB caps F-actin, LatA binds Gactin, and both drugs prevent their polymerization. When the
localization of F-actin was examined using phalloidin staining, it
was abnormally scattered in the cytoplasm of CB-treated 1-cell
embryos, but this was not detected in LatA-treated or in vitro
fertilization-derived control embryos. The spindle was larger in
CB-treated oocytes than in LatA-treated or untreated control
oocytes. LatA treatment also doubled the rate of full-term
development after embryo transfer. These results suggest that
cloning efficiency in mice can be improved by optimizing each
step of the SCNT procedure. Moreover, by using LatA, we could
simplify the procedure with a higher birth rate of cloned mice
compared with our original method.
clone, full term development, histone modifications, nuclear
transfer procedure
INTRODUCTION
Although the successful production of cloned animals by
somatic cell nuclear transfer (SCNT) is a promising technology
with many potential applications in basic research, medicine,
and agriculture [1], the low efficiency of cloning is a major
obstacle to the widespread use of this technology [2]. Since the
first cloned adult mammal was born, many studies of technical
maneuvers have been carried out, such as manipulation of the
1
Supported by Japan Grant-in-Aid for Scientific Research in Priority
Areas 20062015 to T.W.
2
Correspondence: Teruhiko Wakayama, Laboratory for Genomic
Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3
Minatojima-minamimachi, Chuo-ku, Kobe, 650-0046, Japan. E-mail:
[email protected]
3
Correspondence: Yukari Terashita, Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0046, Japan. E-mail:
[email protected]
Received: 25 December 2011.
First decision: 17 January 2012.
Accepted: 29 March 2012.
Ó 2012 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
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cell-cycle stage of donor and recipient cells [3–5], timing of the
activating stimulus [6, 7], careful selection of preferred
recipients [8], aggregation of two cloned embryos [9],
inhibition of cytokinesis [6], and correction of aberrant
epigenetic methylation patterns in DNA or acetylation patterns
in the nuclear histones of cloned embryos [10–12]. In fact, the
cloning success rate was improved significantly by correction
of epigenetic abnormalities with the histone-deacetylase
inhibitor (HDACi) trichostatin A (TSA) [13]. However,
although many agents for controlling epigenetic status have
been trialed in attempts to improve cloning efficiency [14–16],
the generation of live offspring remains inefficient. Recently,
Inoue et al. [17] found that some gene clusters were not
reprogrammed at all by SCNT because of aberrant expression
of the Xist gene and that these abnormalities could be corrected
by injection of Xist-specific short interfering RNA [18]. Thus,
correction of aberrant gene expression of cloned embryos is
important for the production of cloned offspring, but the SCNT
procedure itself also needs more evaluation and refinement. If
some critical negative factors remain in current protocols, it is
important to detect and correct them if we are to increase the
rate of successful full-term clone development.
In most mouse SCNT experiments, cytochalasin B (CB) is
usually used as an F-actin polymerization inhibitor to prevent
the loss of chromosomes into the pseudo-second polar body
(PSPB) [19]. Previously, we reported that the method of
preventing PSPB extrusion had no effect on mouse cloning by
comparing CB, cytochalasin D, and nocodazole [6]. However,
Himaki et al. [20] reported that blastocyst development in
SCNT-generated cloned miniature pig embryos was improved
by using latrunculin A (LatA) instead of CB. LatA, isolated
from the Red Sea sponge Negombata magnifica, was initially
identified as an inhibitor of actin polymerization through its
morphological effects [21]. LatA affects the polymerization of
pure actin by forming a 1:1 molecular complex with G-actin
[22]. In contrast, CB caps the barbed end of actin filaments and
severs them. It sequesters actin monomers or dimers, stimulates
the ATPase activity of G-actin, and blocks all association and
dissociation reactions at that end of the microfilament [23–26].
Although the mechanisms of CB and LatA are slightly
different, both drugs are effective in preventing the loss of
chromosomes into the PSPB after activation. Although Himaki
et al. [20] did not examine the full-term development of
embryos or determine why LatA could increase the blastocyst
formation rate, their data indicate that the prevention of PSPB
extrusion is a critical factor for improving the efficiency of
cloned embryo development. The aim of this study was to
examine the effect of LatA treatment on cloned mouse embryo
development in vivo and in vitro. At first, we evaluated the
optimal concentration of LatA that would inhibit extrusion of
ABSTRACT
TERASHITA ET AL.
oocytes by using 10 mM SrCl2 in Ca2 þ-free CZB medium or in CZB medium
with 2 mM EGTA [30] in the presence of 50 nM TSA and 2.5 or 5 lM LatA for
10 h without changing the medium and then cultured in KSOM until embryo
transfer.
the PSPB. Then, we evaluated the cytoskeletal morphology in
oocytes treated with CB or LatA. Finally, we examined fullterm development of cloned embryos treated with CB or LatA.
In practical terms, mouse SCNT is not only very difficult [7]
but also requires long periods of intensive activity within a
day’s work [27]. Usually, reconstructed oocytes are treated
with CB for more than 5 h after activation to prevent extrusion
of the PSPB, but they must be washed completely within 6 h
because of the toxicity of the drug. These cloned embryos are
then cultured with an HDACi agent for an additional 4 h to
enhance genomic reprogramming and must then be washed
completely again because of the toxicity of the drugs [13].
Thus, although experimentalists might become tired, they must
stay in the laboratory until they complete the last washing step
on the day of mouse SCNT experiments. Therefore, one aim of
this study was to attempt to simplify the mouse SCNT
procedure by using LatA treatment.
Cytoskeleton of Activated Oocytes
Embryos at 0–1 h and 24 h, after being removed from the activation
medium, were fixed in 4% paraformaldehyde in PBS for 30 min, washed twice
with 1% BSA in PBS, transferred into 1% BSA-PBS containing 0.1% Triton X100 (Nacalai Tesque, Kyoto, Japan) and incubated overnight at 48C. Embryos
were then washed twice with 1% BSA-PBS. After that, embryos were
incubated with Alexa Fluor 568-conjugated phalloidin (Molecular Probes,
Eugene OR) for 1 h at room temperature to detect localization of F-actin.
Finally, they were washed with 1% BSA-PBS and observed with a confocal
scanning laser microscope (model FV-1000; Olympus).
Nuclear Transfer
MATERIALS AND METHODS
Animals
B6D2F1 (C57BL/6 3 DBA/2) mice, aged 8–10 weeks, were used to
produce oocytes. Surrogate pseudopregnant females used as embryo transfer
recipients (see below) were ICR strain mice mated with vasectomized males of
the same strain. BD129F1 (B6D2F1 3 129) mice were used as producers from
which cumulus cells were collected as donor cells. B6D2F1 and ICR mice were
purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The
BD129F1 strain was bred in our mouse facility. All animal experiments
conformed to the Guide for the Care and Use of Laboratory Animals and were
approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Center for Developmental Biology.
Collection of Oocytes
In Vitro Fertilization
Mature oocytes were collected from the oviducts of 8- to 10-week-old
female mice that had been induced to superovulate with 5 IU of pregnant mare
serum gonadotropin (Teikokuzoki, Tokyo, Japan) followed by 5 IU of human
chorionic gonadotropin (hCG; Teikokuzoki) 48 h later. Cumulus-oocyte
complexes (COCs) were collected from the oviducts approximately 16 h after
hCG injection. After COCs were collected, they were placed in HEPESbuffered Chatot, Ziomet, and Bavister medium (H-CZB) [28] and treated with
0.1% bovine testicular hyaluronidase (Sigma-Aldrich, St. Louis, MO). After
several minutes, the cumulus-free oocytes were washed twice and then moved
to a droplet of potassium simplex optimized medium (KSOM; Specialty Media
Inc., Philipsburg, NJ) for culture.
For in vitro fertilization (IVF), B6D2F1 spermatozoa from the cauda
epididymidis were cultured in Toyoda Yokoyama Hoshi medium [31] for 1 h at
378C under 5% CO2 in air. Cumulus-intact oocytes were collected and
inseminated with capacitated sperm (final concentration of 50/ll) for 2 h.
Embryo Transfer
Cloned embryos were transferred into the oviducts of pseudopregnant ICR
strain female mice at 0.5 days post coitum, that had been mated with a
vasectomized male the night before transfer. At 19.5 days post coitum, the
offspring were delivered by caesarean section.
Spindle Morphology of Metaphase II Oocytes
Statistical Analysis
Metaphase II (MII) oocytes were treated with CB or LatA for 10 min and
then fixed in 4% paraformaldehyde in PBS. After permeabilization and
blocking, oocytes were incubated with mouse monoclonal anti-b-tubulin (1:100
dilution; BD Biosciences, Franklin Lake, NJ) overnight at 48C, followed by
incubation with Alexa Fluor 488-labeled goat anti-mouse immunoglobulin G
(1:200 dilution; Molecular Probes Inc.) and DAPI (2 lg/ml; Molecular Probes
Inc.). Stained MII oocytes were observed with a confocal scanning laser
microscope (FV-1000 model; Olympus, Tokyo, Japan), and spindle size was
quantified using Fluoview 1.4a software (Olympus) as described previously
[29].
Offspring development rates were evaluated using chi-square tests. Spindle
size was analyzed by ANOVA followed by the Fisher protected least
significant difference test and a P value of ,0.05 was assumed to be
statistically significant.
RESULTS
Optimal Concentration of LatA for Preventing PSPB
Extrusion and Enabling Development after Parthenogenetic
Oocyte Activation
Activation and LatA Treatment
To determine the appropriate concentration of LatA, oocytes were activated
parthenogenetically using 10 mM SrCl2 in Ca2 þ-free CZB medium or in CZB
medium with 2 mM EGTA [30] in the presence of 5 lg/ml CB or 2.5–20 lM
LatA for 6 h and then cultured in KSOM for development.
For SCNT experiments, reconstructed oocytes were activated using 10 mM
SrCl2 in Ca2 þ-free CZB medium in the presence of 50 nM TSA supplemented
with 5 lg/ml CB or 5–10 lM LatA for 6 h. Both CB and LatA were dissolved
in dimethyl sulfoxide to give stock solutions of 500 lg/ml and 1 mM,
respectively. The reconstructed oocytes were then cultured further in KSOM
supplemented with 50 nM TSA for 4 h and then cultured in KSOM until
embryo transfer. In additional experiments, we activated some reconstructed
To determine the effective concentration of LatA for
inhibiting PSPB extrusion and enabling embryo development,
oocytes were activated parthenogenetically with different
concentrations of LatA or CB. As shown in Figure 1,
approximately 60% of parthenogenetic embryos activated with
LatA at 2.5 lM could extrude a PSPB or a 2-cell-like
symmetrical large polar body from the zygote (Fig. 1B), and
these embryos showed lower blastocyst formation rates than
others (Fig. 1C). The minimum concentration of LatA for
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Groups of oocytes were transferred into a droplet of H-CZB containing 5
mg/ml CB on the microscope stage for enucleation of the MII spindle. In
preliminarily experiments, we found that LatA was capable of being used for
enucleation instead of CB. However, those enucleated oocytes needed to be
cultured at 378C under 5% CO2 in air for at least 2 h before nuclear injection,
otherwise many lysed after microinjection. Therefore, in this study, all
enucleation procedures were performed using CB. Oocytes undergoing
microsurgery were held with a holding pipette, and a hole was made in the
zona pellucida following the application of several piezo-pulses (Prime Tech,
Ibaraki, Japan) to an enucleation pipette. The MII chromosome-spindle
complex was aspirated into the pipette with a minimal volume of ooplasm.
After enucleation of all oocytes in one group, they were transferred into
KSOM. For nuclear injection, donor cumulus cells were gently aspirated in and
out of the injection pipette until their nuclei were largely devoid of visible
cytoplasmic membrane. Each nucleus was immediately injected into an
enucleated oocyte. Those reconstructed oocytes were kept in the incubator until
activation.
EFFECTIVE METHOD OF PRODUCING CLONED MICE
dot-like structures of F-actin were also detected in ooplasm 30
min after being washed free of LatA, these disappeared in 30
min. There were no differences between IVF embryos and CBtreated and LatA-treated oocytes 1 h after activation (Fig. 2B,
g–l).
complete prevention of PSPB extrusion and for supporting
blastocyst development was 5 lM.
Effect of CB or LatA on Tubulin Localization in MII Oocytes
To determine the effects of LatA on formation of oocyte MII
spindle, samples of b-tubulin localization were examined.
Although the lengths of spindles treated with CB or LatA were
significantly longer than those of controls, the widths of
spindles in control and LatA-treated oocytes were the same, but
they were significantly different from those in CB-treated
oocytes (P , 0.05; Fig. 2A, Table 1). In addition, 55% of CBtreated oocytes contained a cytoplasmic aster, as shown in
Figure 2A, f, compared with 7% of LatA-treated and 0% of
control oocytes.
Full-Term Development of Cloned Embryos Following CB
or LatA Treatment
To test whether LatA would be effective in improving the in
vivo development of cloned embryos, we transferred cloned
embryos treated with CB or LatA into surrogate mothers.
TABLE 1. Spindle morphology of CB- or LatA-treated MII oocytes.
Effect of CB or LatA on F-Actin Localization of Zygotes from
MII to 2-Cell Stage
Type of oocytes
Control
CB treated
LatA treated
As shown in Figure 2B, F-actin was concentrated in spots
inside the cytoplasm after CB treatment for 6 h (Fig. 2B, b), but
this was never observed in control IVF-derived embryos or in
LatA-treated embryos at 6 h after activation. Although a few
No. of
oocytes
24
25
36
Width
Height
a
25.2 6 2.04
26.6 6 1.78b
24.8 6 1.59a
10.8 6 1.17a
12.5 6 1.12b
12.3 6 0.94b
a,b
Values with different superscripts in the same column are significantly
different (P , 0.05).
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FIG. 1. In vitro development of parthenogenetic embryos treated with CB or LatA. Treatments with 5 lg/ml CB or 2.5–20 lM LatA were used to inhibit
actin polymerization during 6 h of activation. A) The rate of normally activated embryos forming two pronuclei (PN) with no extrusion of the second polar
body (PB) at 6 h after activation is shown. Dark gray bars, 2-PN formation; light gray bars, PB extruded; white bars, 2-cell-like or fragmented embryo. B)
Bright-field images of activated oocytes after CB or LatA treatment for 6 h. Bar ¼ 30 lm. C) Blastocyst formation rates at 96 h were examined. The
minimum concentration of LatA that could be used as an actin polymerization inhibitor instead of CB in the conventional 6 h activation method was 5
lM. All experiments were repeated at least twice and involved at least 60 oocytes. Error bars represent the standard deviation.
TERASHITA ET AL.
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FIG. 2. Effects of CB or LatA treatment on the cytoskeleton. A) MII-stage oocytes were incubated with CB or LatA for 10 min and then fixed. These
treatments did not inhibit spindle formation, but the spindles became larger in CB-treated oocytes. B) IVF-generated control and parthenogenetic embryos
were stained with Alexa Fluor 568-conjugated phalloidin. CB-treated embryos exhibited abnormally scattered F-actin localization during activation, but
this was not observed in IVF-generated or LatA-treated embryos. After 1 h of being washed free of actin polymerization inhibitors, there were no
significant differences between any of the embryo treatments (IVF-generated, CB- or LatA-treated). Bars ¼ 30 lm.
Based on in vitro development data (Fig. 1), we decided to use
5 or 10 lM LatA for this experiment. When 5 lM LatA was
used, the success rate of development to full-term offspring
was increased significantly (9.0%) compared with that of CBtreated cloned embryos (4.5%). Similarly, when 10 lM LatA
was used, the live birth rate was increased to 10.0%, which was
higher than with CB-treated embryos but lower than 5 lM
LatA-treated embryos (Table 2). All offspring survived after
caesarian section and grew normally to more than 8 weeks of
age without any obese phenotype.
Development of a Modified SCNT Procedure Using LatA
In the standard method, activated cloned oocytes are treated
with CB for more than 5 h to prevent extrusion of the PSPB,
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TABLE 2. Full-term development of SCNT-derived embryos treated with CB or LatA.
Cytokinesis
inhibitor
Treatment
duration (h)
No. of
reconstructed
oocytes
No. survived
after Sr
activation
No. of PN
formed (%)a
6
6
6
290
156
112
248
142
90
239 (96.4)
133 (93.7)
78 (86.7)
CB 5 lg/ml
LatA 5.0 lM
LatA 10.0 lM
a
% of offspring from
No. of 2-cell
per PN (%)a
No. of embryos
transferred
(recipients)
No. of live
offspring
Transferred
embryos
Reconstructed
embryos
222 (92.9)
108 (81.2)
60 (76.9)
217 (13)
108 (7)
60 (5)
13
14
6
5.9b
13.0c
10.0
4.5
9.0
5.4
PN, pronuclear stage.
Values with different superscripts in the same column are significantly different (P , 0.05).
b,c
LatA instead of these drugs and found that the method of
inhibiting cytokinesis is an important factor for improving the
success rate of full-term cloning. By altering the method of
preventing PSPB extrusion, the cytoskeleton of cloned
embryos was modified, and the birth rate of cloned mice was
increased. How did LatA improve the cloning success rate? We
have also used a method for preventing PSPB extrusion with
nocodazole [6], which interferes with microtubule assembly
rather than disrupting actin filament polymerization. Although
the spindle disappeared and many small pseudopronuclei were
formed in nocodazole-treated oocytes, the birth rate did not
change significantly between cloned embryos treated with
nocodazole and those treated with CB. Theoretically, nocodazole does not affect F-actin formation but impairs microtubule
assembly, and CB does not affect microtubule assembly but
impairs F-actin formation. Here we confirmed that LatA did
not impair F-actin polymerization or microtubule assembly and
improved the birth rate of cloned mice compared with that with
CB treatment. These results suggest that the formation of
relatively normal F-actin microfilaments and/or interaction
between them might be important for better genomic
reprogramming and birth rates in SCNT procedures.
Actin is an abundant protein present in all eukaryotic cells
and is a major component of the cytoskeleton, which plays
fundamental roles in essential biological processes such as cell
migration, determination of cell shape, and vesicle trafficking.
The polymerization state of actin and the organization of actin
in the cytoplasm are tightly regulated, and both cell features
can respond to extracellular signals [35]. Actin is also a
component of chromatin remodeling complexes involved in
transcriptional activation. Actin binds directly to RNA
polymerases I, II, and III and is required for their full
transcriptional activity [36]. These reports and our data suggest
that the state of F-actin from oocyte activation to PN formation
is vital for nuclear reprogramming and full-term development.
Cloned embryos should have been cultured for 6 h with CB
and TSA and then cultured for another 4 h with TSA to
enhance genomic reprogramming because the most effective
duration of TSA treatment is 10 h [13]. In fact, no cloned
embryos developed to full term when those embryos were
DISCUSSION
In this study, we focused on factors in the SCNT procedure
with the aim of simplifying it and improving cloning
efficiency. Reconstructed oocytes need to be treated with
cytokinesis inhibitors to prevent the loss of somatic cell
chromosomes into the PSPB [7], and CB or cytochalasin D are
now the agents most commonly used [6, 32–34]. Here, we used
TABLE 3. Full-term development of CB- or LatA-treated SCNT embryos.
% of offspring from
Cytokinesis
inhibitor
Treatment
time (h)
No. of
reconstructed
oocytes
Survival
after Sr
activation
No. of PN
formed (%)a
No. of 2-cell
per PN (%)a
No. of embryos
transferred
(recipients)
No. of live
offspring
No. of
transferred
embryos
No. of
reconstructed
embryos
CB 5 lg/ml
LatA 5.0 lM
LatA 5.0 lM
10
10
1 þ 9b
104
157
76
96
144
54
75 (78.1)
132 (91.7)
51 (94.4)
72 (96.0)
122 (92.4)
41 (80.4)
72 (4)
122 (7)
41 (2)
0
19
4
0
15.6
9.8
0
12.1
5.3
a
PN, pronuclear stage.
Reconstructed oocytes were cultured in Ca2þ-free CZB with Sr plus TSA plus LatA for 1 h and then cultured in usual CZB with TSA plus LatA for
another 9 h.
b
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but they must be washed completely within 6 h because of the
toxicity of this drug. Even with a low concentration of CB,
more than 6 h of CB treatment leads to a low rate of blastocyst
formation (Terashita et al., unpublished observations). In fact,
we have shown here that CB damages F-actin (Fig. 2B) and
that no cloned mice were obtained after embryo transfer
following CB treatment for 10 h (Table 3). On the other hand,
the most effective duration of TSA treatment is 10 h from the
beginning of oocyte activation. Therefore, cloned embryos
must be washed twice in the routine SCNT procedure: once at
6 h after activation to remove CB and again at 10 h to remove
TSA. In the present study, we found that LatA caused lower
toxicity than CB, and we surmised that LatA treatment over 6 h
would be feasible. To test the hypothesis that we might be able
to skip one medium change at 6 h after activation by using
LatA instead of CB, we attempted 10-h LatA treatment with
TSA. When reconstructed oocytes were treated with 5 lM
LatA for 10 h, most activated oocytes were prevented from
extruding the PSPB, and the birth rate after embryo transfer
was significantly increased to 15.6% (Table 3). Thus, 10 h of
continuous incubation with LatA was more effective than 6 h
for improving the birth rate of cloned embryos. This also
simplified the SCNT procedure compared with 6-h treatment.
In addition, we considered the effect of culture in Ca2 þ-free
medium for 10 h. SrCl2-induced activation requires Ca2 þ-free
medium [30], but culturing oocytes in Ca2þ-free medium for a
long time might impair embryo development. However, there
was no significant difference in the final birth rate between
oocytes treated with 1-h or 10-h culture in Ca2 þ-free medium.
Thus, extended oocyte culture in Ca2þ-free medium did not
have significant detrimental effects.
TERASHITA ET AL.
ACKNOWLEDGMENT
We thank M. Tokoro, Y. Sakaide, and T. Oyanagi for assistance in
preparing the manuscript. We are grateful to the Laboratory for Animal
Resources and Genetic Engineering for housing the mice.
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treated with CB for 10 h. Here, we showed that the toxicity of
LatA was much lower than that of CB, and we succeeded in
producing many cloned mice without washing the embryos at
the 6-h time point after activation. Simply replacing CB with
LatA in the conventional method led to a higher success rate,
but the most effective method was 10 h continuous treatment
with LatA. This procedure allowed the experimenter to skip the
stage of changing the medium at 6 h and might reduce harm.
Results of the present study provide a more effective method
for SCNT cloning but do not explain how F-actin polymerization affects the developmental ability of cloned embryos.
Further work will be required to clarify some issues relating to
F-actin and reprogramming efficiency at the pronuclear stage.
Although this new method allows us to skip one step of the
SCNT procedure (washing embryos at 6 h after activation), we
hope that this simplified method of activation will help cloning
researchers to save time because the SCNT protocol is very
difficult and requires many hours of experiment and practice.