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Somatic cell nuclear transfer (SCNT) is a very important reproductive technology with many diverse applications, such as fast multiplication of elite animals, the production of transgenic animals and embryonic stem (ES) cells. However, low cloning efficiency, a low live birth rate and the abnormally high incidence of abnormalities in the offspring born are attributed to incomplete or aberrant nuclear reprogramming. In SCNT embryos, the aberrant expression pattern of the genes throughout embryonic development is responsible for the incomplete nuclear reprogramming. The present study was carried out to identify the differential gene expression (DEGs) profile and molecular pathways of the SCNT and IVF embryos at different developmental stages (2 cell, 8 cell and blastocyst stages). In the present study, 1164 (2 cell), 1004 (8 cell) and 530 (blastocyst stage) DEGs were identified in the SCNT embryos as compared to IVF embryos. In addition, several genes such as ZEB1, GDF1, HSF5, PDE3B, VIM, TNNC, HSD3B1, TAGLN, ITGA4 and AGMAT were affecting the development of SCNT embryos as compared to IVF embryos. Further, Gene Ontology (GO) and molecular pathways analysis suggested, SCNT embryos exhibit variations compared to their IVF counterparts and affected the development of embryos throughout the different developmental stages. Apart from this, q-PCR analysis of the GDF1, TMEM114, and IGSF22 genes were utilized to validate the RNA-seq data. These findings contribute valuable insights about the different genes and molecular pathways underlying SCNT embryo development and offer crucial information for improving SCNT efficiency.

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Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.

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Despite the success of cloning technology in the production of offspring across several species, its application on a wide scale is severely limited by the very low offspring rate obtained with cloned embryos. The expression profile of microRNAs (miRNAs) in cloned embryos throughout embryonic development is reported to deviate from regular patterns. The present study is aimed at determining the dynamics of the global expression of miRNA profile in cloned and in-vitro fertilization (IVF) pre-implantation embryos at different developmental stages, i.e., the two-cell, eight-cell, and blastocyst stages, using next-generation sequencing. The results of this study suggest that there is a profound difference in global miRNA profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. The cloned embryos differ from their IVF counterparts in enriched Gene Ontology (GO) terms of biological process, molecular function, cellular component, and protein class categories in terms of the targets of differentially expressed miRNAs. The major pathways related to embryonic development, such as the Wnt signaling pathway, the apoptosis signaling pathway, the FGF signaling pathway, the p53 pathway, etc., were found to be affected in cloned relative to IVF embryos. Overall, these data reveal the distinct miRNA profile of cloned relative to IVF embryos, suggesting that the molecules or pathways affected may play an important role in cloned embryo development.

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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.

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Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

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Islam is a religion that inspires its followers to seek knowledge continually and nurtures innovation, within the realms of Islamic rulings, towards an ameliorated quality of life. Up-to-date biotechnological techniques, specifically animal cloning, are involved in advancing society's health, social, and economic domains. The goal of animal cloning includes the production of genetically modified animal for human consumption. Therefore, this research endeavoured to study animal cloning's current scientific findings, examine the by-product of said process, and determine its permissibility in an Islamic context. This study employed descriptive literature reviews. Results concluded that animal cloning, especially in mammals, does not occur naturally as in plants. A broadly trusted and efficient animal cloning method is known as Somatic Cell Nuclear Transfer (SCNT), which includes three principal steps: oocyte enucleation; implantation of donor cells (or nucleus); and the activation of the embryo. Nevertheless, the limitations of SCNT, particularly to the Large Offspring Syndrome (LOS), should be noted. One of the forms of the application of animal cloning is in agriculture. From an Islamic perspective, determining the permissibility of consuming cloned animals as food is essentially based on whether the cloned animal conforms to Islamic law's principles and criteria. Islam interdicts animal cloning when it is executed without benefiting humans, religion, or society. Nonetheless, if it is done to preserve the livelihood and the needs of a community, then the process is deemed necessary and should be administered following the conditions outlined in Islam. Hence, the Islamic ruling for animal cloning is not rigid and varies proportionately with the current fatwa.

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Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

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While B cells play a significant role in the onset of type-1 diabetes (T1D), little is know about their role in those early stages. Thus, to gain new insights into the role of B cells in T1D, we converted a physiological early pancreas-infiltrating B cell into a novel BCR mouse model using Somatic Cell Nuclear Transfer (SCNT). Strikingly, SCNT-derived B1411 model displayed neither developmental block nor anergy. Instead, B1411 underwent spontaneous germinal center reactions. Without T cell help, B1411-Rag1-/- was capable of forming peri-/intra-pancreatic lymph nodes, and undergoing class-switching. RNA-Seq analysis identified 93 differentially expressed genes in B1411 compared to WT B cells, including Irf7, Usp18, and Mda5 that had been linked to a potential viral etiology of T1D. We also found various members of the oligoadenylate synthase (OAS) family to be enriched in B1411, such as Oas1, which had recently also been linked to T1D. Strikingly, when challenged with glucose B1411-Rag1-/- mice displayed impaired glucose tolerance.

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Xist is an X-linked ribonucleic acid (RNA) gene responsible for the cis induction of X chromosome inactivation (XCI). In cloned mammalian embryos, Xist is ectopically activated at the morula to blastocyst stage on the X chromosome that is supposed to be active, thus resulting in abnormal XCI. Suppression of erroneous Xist expression by injecting small interfering RNA (siRNA) remarkably increased the developmental efficiency of cloned male mouse embryos by approximately 10-fold. However, injection of anti-Xist siRNA resulted in only a slight increase in the developmental ability of injected cloned male pig embryos because the blocking effect of the injected siRNA was not maintained beyond the morula stage, which is 5 days post-activation. To develop a more effective approach for suppressing the ectopic expression of Xist in cloned pig embryos, we compared the silencing effect of short hairpin RNA (shRNA) and siRNA on Xist expression and the effects of these two Xist knockdown methods on the developmental competence of cloned male pig embryos. Results indicated that an shRNA-based RNA interference (RNAi) has a longer blocking effect on Xist expression than an siRNA-mediated RNAi. Injection of anti-Xist shRNA plasmid into two-cell-stage cloned male pig embryos effectively suppressed Xist expression, rescued XCI at the blastocyst stage, and improved the in vitro developmental ability of injected cloned embryos. These positive effects, however, were not observed in cloned male pig embryos injected with anti-Xist siRNA. This study demonstrates that vector-based rather than siRNA-mediated RNAi of Xist expression can be employed to improve pig cloning efficiency.

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The nuclear receptor corepressor Hairless (HR) interacts with nuclear receptors and controls expression of specific target genes involved in hair morphogenesis and hair follicle cycling. Patients with HR gene mutations exhibit atrichia, and in rare cases, immunodeficiency. Pigs with HR gene mutations may provide a useful model for developing therapeutic strategies because pigs are highly similar to humans in terms of anatomy, genetics, and physiology. The present study aimed to knockout the HR gene in pigs using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated-9 (Cas9) system and to investigate the molecular and structural alterations in the skin and thymus. We introduced a biallelic mutation into the HR gene in porcine fetal fibroblasts and generated nine piglets via somatic cell nuclear transfer. These piglets exhibited a lack of hair on the eyelids, abnormalities in the thymus and peripheral blood, and altered expression of several signaling factors regulated by HR. Our results indicate that introduction of the biallelic mutation successfully knocked out the HR gene, resulting in several molecular and structural changes in the skin and thymus. These pigs will provide a useful model for studying human hair disorders associated with HR gene mutations and the underlying molecular mechanisms.

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As a natural plant-derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 µM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) gene and significant downregulation of the pro-apoptotic BCL2-associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 µM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 µM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.

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Oocyte quality, which is directly related to reprogramming competence, is a major important limiting factor in animal cloning efficiency. Compared with oocytes matured in vivo, in vitro matured oocytes exhibit lower oocyte quality and reprogramming competence primarily because of their higher levels of reactive oxygen species. In this study, we investigate whether supplementing the oocyte maturation medium with melatonin, a free radical scavenger, could improve oocyte quality and reprogramming competence. We found that 10-9 M melatonin effectively alleviated oxidative stress, markedly decreased early apoptosis levels, recovered the integrity of mitochondria, ameliorated the spindle assembly and chromosome alignment in oocytes, and significantly promoted subsequent cloned embryo development in vitro. We also analyzed the effects of melatonin on epigenetic modifications in bovine oocytes. Melatonin increased the global H3K9 acetylation levels, reduced the H3K9 methylation levels, and minimally affected DNA methylation and hydroxymethylation. Genome-wide expression analysis of genes in melatonin-treated and nontreated oocytes was also conducted by high-throughput RNA sequencing. Our results indicated that melatonin ameliorates oocyte oxidative stress and improves subsequent in vitro development of bovine cloned embryos.

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Forced expression of human telomerase reverse transcriptase (hTERT) has been used to immortalize mammalian cells. Here, we report conditional extension of proliferative lifespan of adult sheep somatic cells by introducing tetracycline-inducible (Tet-on) expression of hTERT. After transfecting adult sheep fibroblasts with the vector for Tet-on induced conditional expression of hTERT, we obtained several hTERT-positive clones that exhibited extended-lifespan under the induction of tetracycline analogue, doxycycline. Further assays for a representative cell clone A3h38 indicated that the cells had a much stronger proliferative ability than control primary cells, as assessed by population doubling levels and single-cell cloning efficiency. A3h38 cells could maintain vigorous growth in culture for more than 150 days but they became senescent when hTERT expression was abrogated by withdrawal of doxycycline. Although having undergone long-term culture, the nuclei of A3h38 cells could support higher preimplantation development of somatic cell nuclear transfer embryos than control primary cells. These results demonstrated that conditional expression of hTERT could reversibly extend the lifespan of adult sheep fibroblasts, enhance their proliferation and maintain their ability to be reprogrammed after nuclear transfer. This strategy would also be applicable to many other somatic cell types in more species.

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Age reprogramming represents a novel method for generating patient-specific tissues for transplantation. It bypasses the de-differentiation/redifferentiation cycle that is characteristic of the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell technologies that drive current interest in regenerative medicine. Despite the obvious potential of iPS and NT-ES cell-based therapies, there are several problems that must be overcome before these therapies are safe and routine. As an alternative, age reprogramming aims to rejuvenate the specialized functions of an old cell without de-differentiation; age reprogramming does not require developmental reprogramming through an embryonic stage, unlike the iPS and NT-ES cell-based therapies. Tests of age reprogramming have largely focused on one aspect, the epigenome. Epigenetic rejuvenation has been achieved in vitro in the absence of de-differentiation using iPS cell reprogramming factors. Studies on the dynamics of epigenetic age (eAge) reprogramming have demonstrated that the separation of eAge from developmental reprogramming can be explained largely by their different kinetics. Age reprogramming has also been achieved in vivo and shown to increase lifespan in a premature ageing mouse model. We conclude that age and developmental reprogramming can be disentangled and regulated independently in vitro and in vivo.

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Epigenetic reprogramming and somatic cell nuclear transfer (SCNT) cloning efficiency were recently enhanced using histone deacetylase inhibitors (HDACis). In this study, we investigated the time effect of CI994, an HDACi, on the blastocyst formation rate, acetylation levels of H3K9 and H4K12, DNA methylation levels of anti-5-methylcytosine (5mC), and some mRNA expression of pluripotency-related genes in pig SCNT embryos. Treatment with 10 µM CI994 for 24 hours significantly improved the blastocyst formation rate of SCNT embryos in comparison with the untreated group (p < 0.05). Moreover, average fluorescence intensities of H3K9 and H4K12 in CI994-treated embryos were remarkably increased at the pseudo-pronuclear stage, but not at the blastocyst stage. The intensity of POU5F1 was higher in CI994-treated blastocysts than in control blastocysts, whereas that of 5mC did not differ between the two groups. The percentage of apoptotic cells in blastocysts was significantly higher in the untreated group than in the CI994-treated group. mRNA levels of POU5F1 and SOX2 were significantly increased in the CI994-treated group. These observations suggest that optimum exposure (10 µM for 24 hours) to CI994 after activation elevates the level of histone acetylation and subsequently improves the in vitro development of pig SCNT embryos.

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MicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with in vitro fertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

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The success of cloned animal "Dolly Sheep" demonstrated the somatic cell nuclear transfer (SCNT) technique holds huge potentials for mammalian asexual reproduction. However, the extremely poor development of SCNT embryos indicates their molecular mechanism remain largely unexplored. Deciphering the spatiotemporal patterns of gene expression in SCNT embryos is a crucial step toward understanding the mechanisms associated with nuclear reprogramming. In this study, a valuable transcriptome recourse of SCNT embryos was firstly established, which derived from different inter-/intra donor cells. The gene co-expression analysis identified 26 cell-specific modules, and a series of regulatory pathways related to reprogramming barriers were further enriched. Compared to the intra-SCNT embryos, the inter-SCNT embryos underwent only complete partially reprogramming. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and mediators were incomplete activated in inter-SCNT embryos. The inter-SCNT embryos only wasted the stored maternal mRNA of master regulators, but failed to activate their self-sustained pathway of RNA polymerases. The KDM family of epigenetic regulator also seriously delayed in inter-SCNT embryo reprogramming process. Our study provided new insight into understanding of the mechanisms of nuclear reprogramming.

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OBJECTIVE: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). METHODS: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. RESULTS: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. CONCLUSION: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

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Cloning remains as an important technique to enhance the reconstitution and distribution of animal population with high-genetic merit. One of the major detrimental factors of this technique is the abnormal epigenetic modifications. MGCD0103 is known as a histone deacetylase inhibitor. In this study, we investigated the effect of MGCD0103 on the in vitro blastocyst formation rate in porcine somatic cell nuclear transferred (SCNT) embryos and expression in acetylation of the histone H3 lysine 9 and histone H4 lysine 12. We compared the in vitro embryonic development of SCNT embryos treated with different concentrations of MGCD0103 for 24 hours. Our results reported that treating with 0.2-µM MGCD0103 for 24 hours effectively improved the development of SCNT embryos, in comparison to the control group (blastocyst formation rate, 25.5 vs. 10.7%, P < 0.05). Then we tested the in vitro development of SCNT embryos treated with 0.2-µM MGCD0103 for various intervals after activation. Treatment for 6 hours significantly improved the development of pig SCNT embryos, compared with the control group (blastocyst formation rate, 21.2 vs. 10.5%, P < 0.05). Furthermore, MGCD0103 supplementation significantly (P < 0.05) increases the average fluorescence intensity of AcH3K9 and AcH4K12 in embryos at the pseudo-pronuclear stage. To examine the in vivo development, MGCD0103-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and three fetuses developed. These results suggest that MGCD0103 can enhance the nuclear reprogramming and improve in vitro developmental potential of porcine SCNT embryos.

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