Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos

2020 ◽  
Vol 32 (5) ◽  
pp. 508
Author(s):  
S. Sah ◽  
A. K. Sharma ◽  
S. K. Singla ◽  
M. K. Singh ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Cell Stage ◽  
Morula Stage

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

185 DEVELOPMENT CAPACITY OF PRE- AND POSTPUBERTAL PIG OOCYTES EVALUATED BY SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETIC ACTIVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 241 ◽  
Author(s):  
H. S. Pedersen ◽  
R. Li ◽  
Y. Liu ◽  
P. Løvendahl ◽  
P. Holm ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Time Interval ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Development Capacity ◽  
Developmental Capacity

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible perivitelline space) or bad before used for PA (good and bad) or SCNT (good). The PA and SCNT were performed as before with minor modifications (Cryobiol. 64, 60; Cell. Reprogr. 13, 521) before culture for 6 days in a standard or timelapse incubator. Rates of cleavage (CL%, Day 2), blastocyst (BL%, Day 6), and blastocyst cell number (Hoechst 33342) were recorded. For PA embryos in a timelapse incubator (26 oocytes/group; 2 replicates), the first appearance of 2-cell stage was recorded. Between groups, CL% and BL% were analysed by chi-square and cell number by t-test. Results are presented in the table for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences in CL% (90–96) and blastocyst cell number (51–59) between groups. The BL% was higher in medium gilts and sows (41; 45) compared with large gilts (21). The BL% of bad oocytes was 1% from all 4 groups (176 oocytes, 25 replicates). Time interval for appearance of 2-cell stage for embryos developing into blastocysts showed no differences between groups (19–20 h). Within groups, this time interval showed a larger standard deviation for embryos not developing v. embryos developing into blastocysts. It is concluded that (a) sow oocytes have higher developmental capacity compared to gilts, (b) small gilt oocytes are not developmentally competent, (c) measurement of inside-ZP diameter, combined with morphological selection, is useful to remove non-competent oocytes. Further studies are needed to dissect the developmental capacity of medium and large gilt oocytes. Also, further timelapse studies may reveal a time interval in which the first cleavage of embryos with high developmental capacity takes place. Table 1.Rates of cleavage (CL%), blastocyst (BL%), and total no. of cells (mean ± SEM) in blastocysts of PA embryos from gilts and sows1

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer

2015 ◽  
Vol 27 (3) ◽  
pp. 544 ◽  
Author(s):  
H. S. Pedersen ◽  
Y. Liu ◽  
R. Li ◽  
S. Purup ◽  
P. Løvendahl ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Oocyte Growth ◽  
Parthenogenetic Activation ◽  
Selection Of

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter (‘small’ ≤110 µm; ‘medium’ >110 µm; ‘large’ ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer

2003 ◽  
Vol 15 (3) ◽  
pp. 179 ◽  
Author(s):  
Goo Jang ◽  
Byeong Chun Lee ◽  
Sung Keun Kang ◽  
Woo Suk Hwang
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Fertilization ◽  
Chondroitin Sulfate ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Developmental Competence ◽  
Vitro Fertilization

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.

scholarly journals 71 DEVELOPMENTAL DELAY OF PRE-IMPLANTATION OVINE IN VITRO CULTURED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 185
Author(s):  
P. Tveden-Nyborg ◽  
T. Peura ◽  
K. Hartwich ◽  
P. Maddox-Hyttel
Keyword(s):  
Somatic Cell ◽  
Developmental Delay ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Germ Layers ◽  
Visceral Mesoderm

Despite advances in the production of somatic cell nuclear transfer (SCNT) embryos, significant embryo losses are persistent, particularly around implantation. Malformations of the placenta and in a variety of organs are seen, and have been linked to deviant epigenetic reprogramming. The aim of the present study was to examine the formation of germ layers, which are prerequisites for formation of the embryo proper and placenta, in invivo-derived (in vivo), partly in vitro-cultured (IVC), and SCNT ovine embryos. Embryos were derived as follows: In vivo embryos (n = 27) were flushed from the uterus on Days 7, 9, 11, and 13. For IVC embryos (n = 22) in vivo zygotes were flushed, followed by culture in the presence of 20% human serum, transfer to the uterus on Day 6, and flushing as in vivo embryos. SCNT embryos (n = 41) were produced by fusion of serum starved granulosa cells with enucleated oocytes, followed by activation, culture in SOF, transfer to the uterus on Day 6, and flushing as described for in vivo embryos. Recovered embryos were processed for light microscopy (LM) and transmission electron microscopy (TEM), and paraffin sections were immunohistochemically labelled for the germ layers: alpha-1-fetoprotein for potential endoderm, cytokeratin-8 for potential ectoderm, and vimentin for potential mesoderm. A consistent delay of the IVC and particularly the SCNT embryos was noted throughout all time points: On Days 7 and 9, differentiation of the inner cell mass into hypoblast and epiblast was evident in 7 out of 12 in vivo embryos, whereas this phenomenon was less prominent or absent in 9 out of 13 IVC and 13 out of 15 SCNT embryos. Furthermore, 6 of the IVC and 12 of the SCNT embryos lacked an identifiable embryonic disc. On Day 11, half of the in vivo embryos had initiated gastrulation, evidenced by localization of endoderm and mesoderm precursor cells between the hypoblast and the epiblast. This feature was noted in only a single IVC and in none of the SCNT embryos. On Day 13, all in vivo embryos had completed gastrulation including the formation of somatic and visceral mesoderm. This feature was noted in only 1 out of 3 IVC and in none of the SCNT embryos. Likewise, amniotic folds were seen in one third of the in vivo embryos at this stage, but not observed in any IVC or SCNT embryos. The immunohistochemical markers displayed the same cell lineage localization in all three groups of embryos, but a developmental delay in the IVC and in particular the SCNT embryos was evident. In conclusion, ovine IVC and SCNT embryos develop at a slower rate than in vivo embryos at least up until Day 13 of gestation.

332 EMBRYONIC DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER OF PORCINE OOCYTES MEIOTICALLY INHIBITED WITH ROSCOVITINE

2006 ◽  
Vol 18 (2) ◽  
pp. 273
Author(s):  
S. W. Kim ◽  
D. H. Kim ◽  
J. S. Seo ◽  
G. S. Im ◽  
B. C. Yang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Apoptotic Cell ◽  
Treated Group ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Maturation Medium

Numerous factors affect on the developmental competence of cloned embryos, and one of the factors might be the disturbed synchronization of nuclear and cytoplasm maturation. Roscovitine, a purine known to specifically inhibit M-phase promoting factor (MPF) kinase activity by blocking the ATP in numerous cell systems, has been successfully used in maintaining porcine oocytes at GV stage without affecting their developmental potential. However, developmental ability of roscovitine treated porcine oocytes after nuclear transfer has not been evaluated. The purpose of this study was to examine the development of nuclear transferred porcine embryos after meiotic inhibition with roscovitine (ROS). Cumulus-oocyte complexes (COCs) were collected from antral follicles of slaughtered prepubertal gilts. COCs were cultured in pre-maturation medium (TCM-199 containing 50 �M Roscovitine) for 24 h, and then further cultured in conventional maturation medium for 44 h. A control group was cultured in the maturation medium for 44 h. Matured oocytes were enucleated and a porcine fetus cell was inserted into each enucleated oocyte. Couplets were simultaneously fused and activated with electric pulse of two 1.2 kV/cm for 30 �s. Nuclear transferred (NT) embryos were cultured in PZM-1 medium for 6 days (five replicates). Apoptotic cell death was analyzed by using a TUNEL assay and total cell number was examined by Hoechest 33342 counterstaining. At 3 h after fusion, NT embryos were fixed for microfilament staining. Data were analyzed by ANOVA and Student's t-test. The rates of fusion, cleavage, and blastocyst formation of the ROS-treated group (85, 68, and 18%, respectively) after nuclear transfer did not differ from control (78, 76, and 16%, respectively). The cell number in blastocysts of the ROS-treated group (30.8 � 10.6) was significantly lower than that of the control (42.3 � 13.7) (P < 0.01), but the mean proportion of apoptotic cells was not different between the two groups (6.9 � 7.1 and 4.8 � 4.9% for control and ROS group, respectively). Recovery of microfilaments after fusion was delayed in NT embryos derived from ROS-treated oocytes. This study demonstrated that porcine oocytes pre-cultured for 24 h in presence of roscovitine can be developed to blastocysts after somatic cell nuclear transfer. This could provide flexibility for studying porcine oocyte development and embryo cloning.

51 DELAYED NUCLEOLOGENESIS IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2010 ◽  
Vol 22 (1) ◽  
pp. 183
Author(s):  
R. S. Deshmukh ◽  
O. Østrup ◽  
E. Lemme ◽  
B. Peterson ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Condensed Chromatin ◽  
Preimplantation Embryos ◽  
Metaphase Chromosomes ◽  
Cell Stage

Nucleolus is known to be a well-suited morphological marker for embryo technologies. Failure in de novo nucleolar formation during embryonic genome activation (EGA) has been observed in many species. The aim of the present study was to investigate nuclear changes and nucleolar formation during EGA in the porcine preimplantation embryos developed in vivo and produced by somatic cell nuclear transfer (SCNT). Embryos were collected at early and late 1-cell stage, 2-, 4-, and 8-cell stage, early and late blastocyst stage, fixed in 3% glutaraldehyde for 1 h, and processed for transmission electron microscopy. In vivo embryos from 1- and 2-cell stages showed electron dense, spherical nucleolar precursor bodies (NPB) in centrally located nuclei with well-developed nuclear envelope and condensed chromatin. Two 1-cell-stage embryos, however, had represented metaphase chromosomes in the periphery. At the 4-cell stage, in vivo embryos displayed fibrillo-granular nucleoli containing all 3 functional nucleolar compartments: fibrillar centers (FC), dense fibrillar component (DFC), and granular component (GC). The nuclei were centrally located, round, and had complete nuclear envelopes. The same types of nuclei and nucleoli were observed for all following stages. On the other hand, embryos produced by SCNT at early 1-cell stage showed centrally located, irregular-shaped nuclei with incomplete nuclear envelopes and condensed chromatin with large intact NPB. Exceptionally, 1 out of the 5 embryos presented a peripheral nucleus with partially condensed chromatin lacking nuclear envelope and fibrillo-granular nucleolus probably persisting from donor fibroblast. Only 2 out of 5 late-1-cell SCNT embryos showed nuclear structures. The nuclei had irregular shapes, complete nuclear membranes, and contained large NPB. At the 2- and 4-cell stages, the embryos presented central nuclei with complete nuclear envelopes. Some of the embryos showed more than one nucleus of varying shapes and sizes. The fibrillo-granular nucleoli were first observed toward the 8-cell stage. The embryos from this stage contained irregularly shaped nuclei with well-developed nuclear envelopes. The nucleoli displayed fibrillar and granular compartments in SCNT 8-cell stage embryos, but lacked the typically structured functional nucleoli observed in in vivo embryos. The absence of formation of functional nucleoli at the 4-cell stage and altered nuclear ultrastructure during the EGA in SCNT embryos, thus, may be one of the main reasons for decreased developmental competence of SCNT embryos.

70 FIBROBLAST GROWTH FACTOR 4 PROMOTES THE DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN MICE

2010 ◽  
Vol 22 (1) ◽  
pp. 193
Author(s):  
T. Mitani ◽  
M. Morita ◽  
M. Anzai ◽  
Y. Nishiyama ◽  
K. Moriki ◽  
...  
Keyword(s):  
Growth Factor ◽  
Somatic Cell ◽  
Fibroblast Growth Factor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Preimplantation Embryos ◽  
Fibroblast Growth

Somatic cell nuclear transfer (SCNT) embryos can develop during the preimplantation period; however, most of these die after implantation period. A transcription factor, Cdx2, promotes differentiation of extraembryonic tissues and appears to be involved in the segregation of inner cell mass (ICM) and trophectoderm (TE) in preimplantation embryos. So far, we have demonstrated that the expression of Cdx2 in mouse SCNT embryos is delayed and its expression level is significantly lower than that in intracytoplasmic sperm injection (ICSI) embryos. Moreover, the ectopic expression of Oct-3/4 was observed in the TE tissues of SCNT blastocysts, but not in ICSI blastocysts. Fibroblast growth factor (FGF) receptor 2 (FGFR2) is specifically expressed in 8-cell to morula-stage embryos and trophectoderm (TE) and is essential for implantation; however, FGFR2 expression in SCNT embryos significantly decreases compared with IVF embryos. Therefore, it is likely that abnormality of differentiation that is controlled in development of pre-implantation in SCNT embryos leads to a rapid decrease of subsequent developmental ability. Then, we investigated the effects of FGF4 on development of SCNT embryos. Mouse SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998). B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. Data were analyzed by Student’s t-test. First, the timing to start adding FGF4 was decided by FGFR2 expression time about 54 h after cell injection and treated for 3, 6, 12, 24, and 42 h thereafter. In the case of FGF4 concentration at 25 ng mL-1 with treating time of 6 h from the 4- to 8-cell stages, SCNT embryos significantly promoted the development to morula and blastocyst stages (91 and 45%, respectively) compared with IVF embryos (80 and 30%, respectively; P < 0.05). However, longer treatment of 42 h with FGF4 made their morphology considerably worse. Then, concentrations of FGF4 at 5, 25, 50, 250, and 500 ng mL-1 with treating time of 6 h was examined. In case of FGF4 concentration at 25 and 50 ng mL-1, SCNT embryos significantly promoted the development to morula and blastocyst stages (P < 0.05). Immunohistochemical analysis showed segregation of the expression of Oct-3/4 and Cdx2 in ICM and TE, respectively, in FGF4-treated SCNT embryos, unlike in the case of nontreated SCNT embryos, which showed an ectopic expression of Oct-3/4 in TE tissues. Furthermore, after the transplantation of SCNT embryos treated with FGF4 at 50 ng mL-1 and the treating time of 6 h to recipient mice, most of the transferred embryos implanted and cloned mice were successfully produced as well as nontreated SCNT embryos. Therefore, FGF4 facilitates the development of SCNT embryos especially to the morula and blastocyst stages. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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