Mammalian Somatic Cell Nuclear Transfer Research Articles

Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years.

Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.

BESC from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells.

Epigenetic reprogramming after mammalian somatic cell nuclear transfer is often incomplete, resulting in low efficiency of cloning. However, gene expression and histone modification analysis indicated high similarities in transcriptome and epigenomes of bovine embryonic stem cells from in vitro fertilized and somatic cell nuclear transfer embryos. Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock, they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently derived from bovine blastocysts, a detailed characterization of their molecular state is lacking. Here, we apply cutting-edge technologies to analyze the transcriptomic and epigenomic landscape of bovine ESC (bESC) obtained from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. bESC were efficiently derived from SCNT and IVF embryos and expressed pluripotency markers while retaining genome stability. Transcriptome analysis revealed that only 46 genes were differentially expressed between IVF- and SCNT-derived bESC, which did not reflect significant deviation in cellular function. Interrogating histone 3 lysine 4 trimethylation, histone 3 lysine 9 trimethylation, and histone 3 lysine 27 trimethylation with cleavage under targets and tagmentation, we found that the epigenomes of both bESC groups were virtually indistinguishable. Minor epigenetic differences were randomly distributed throughout the genome and were not associated with differentially expressed or developmentally important genes. Finally, the categorization of genomic regions according to their combined histone mark signal demonstrated that all bESC shared the same epigenomic signatures, especially at gene promoters. Overall, we conclude that bESC derived from SCNT and IVF embryos are transcriptomically and epigenetically analogous, allowing for the production of an unlimited source of pluripotent cells from high genetic merit organisms without resorting to transgene-based techniques.

Effect of serum starvation and contact inhibition on dermal fibroblast cell cycle synchronization in two species of wild felids and domestic cat

Abstract Cell cycle synchronization of donor cells is an important step in mammalian somatic cell nuclear transfer (SCNT). This study was designed to compare the efficiency of serum starvation (Ss) and contact inhibition (cI) on cell cycle synchronization of jaguarundi, manul, and domestic cat skin fibroblasts, in the production of G0/G1 cells suitable for SCNT in felids. Ss was performed after the growing (G) cells reached 40–50% (G50+Ss), 60–70% (G70+Ss) and full confluency (Fc), i.e. in association with cI (cI+Ss). Frozen-thawed cells were cultured to the given state of confluency (d0; controls), and subjected to Ss or cI for 1, 3, and 5 days (d). In manul, the effect of Ss on arresting fibroblasts in the G0/G1 phase was noted after just 1d of culture at G70 confluence, while G50+Ss and cI+Ss were effective after 5d of treatment. In jaguarundi, 1–5d of G50+Ss and 5d of G70+Ss increased the percentage of G0/G1 cells versus d0 (P<0.01), with 5d of G70+Ss producing more (P<0.05) quiescent cells than after the same period of G50+Ss, cI+Ss and cI. In the domestic cat, Ss was efficient only after 3 and 5d of G50+Ss. In all species, cI alone failed to increase the proportion of G0/G1 cells compared to d0, however in the domestic cat, 5d of cI was more efficient than the same period of G50+Ss. In jaguarundi, >93% of cells were already in G0/G1 phase at d0 of Fc, suggesting that culture to Fc could be also a valuable method for fibroblast cell cycle synchronization in this species. In contrast to cI, prolonged Ss generated cell loss and could induce apoptosis and/or necrosis. In conclusion, Ss was the more efficient method for skin fibroblast cell cycle synchronization at the G0/G1 phase in manul, jaguarundi and the domestic cat. The response of cells to the treatments was species-specific, depending on cell confluence and duration of culture. This research may find application in preparing donor karyoplasts for SCNT in felids.

Characterization of transcriptional activity during ZGA in mammalian SCNT embryo†.

Developmental arrest of somatic cell nuclear transfer (SCNT) embryos first occurs at zygotic/embryonic genome activation (ZGA/EGA), which is critical for preimplantation development. However, study on transcriptome of SCNT embryos during ZGA/EGA is limited. In the present study, we performed RNA sequencing (RNA-seq) of the eight-cell SCNT embryos in goat and provide cross-species analysis of transcriptional activity of SCNT embryos during ZGA/EGA in mice, human, bovine, and goat. RNA-seq data revealed 3966 differentially expressed genes (DEGs) failed to be reprogrammed or activated during EGA of SCNT embryos in goat. Series test of cluster analysis showed four clusters of DEGs and similar changes of the clusters in the four species. Specifically, genes in cluster 3 were somehow upregulated compared with the donor cells and the in vitro fertilization embryo. Moreover, the histone methylation key players and N6-methyladenosine modifiers (SUV39H1, SETDB1, SETD2, KDM5B, IGF2BP1, and YTHDF2) were differentially expressed in SCNT embryos of all species. Finally, we identified three modules correlated with the development of SCNT embryos in mice and screened 288 genes (such as BTG4, WEE1, KLF3, and USP21) that are likely critical for SCNT reprogramming using weighted gene correlation network analysis. Our data will broaden the current understanding of transcriptome activity during stochastic reprogramming events and provide an excellent source for future studies.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

How to improve mouse cloning

The mouse is the most extensively used mammalian laboratory species in biology and medicine because of the ready availability of a wide variety of defined genetic and gene-modified strains and abundant genetic information. Its small size and rapid generation turnover are also advantages compared with other experimental animals. Using these advantages, somatic cell nuclear transfer (SCNT) in mice has provided invaluable information on epigenetics related to SCNT technology and cloning, playing a leading role in relevant technical improvements. These improvements include treatment with histone deacetylase inhibitors, correction of Xist gene expression (controlling X chromosome inactivation), and removal of methylated histones from SCNT-generated embryos, which have proven to be effective for SCNT cloning of other species. However, even with the best combination of these treatments, the birth rate in cloned offspring is still lower than intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). One remaining issue associated with SCNT is placental enlargement (hyperplasia) found in late pregnancy, but this abnormality might not be a major cause for the low efficiency of SCNT because many SCNT-derived embryos die before their placentas start to enlarge at midgestation (early postimplantation stage). It is known that, at this stage, undifferentiated trophoblast cells in the extraembryonic tissue of SCNT-derived embryos fail to proliferate. Understanding the molecular mechanisms is essential for further technical improvements of mouse SCNT, which might also provide clues for technical breakthroughs in mammalian SCNT and cloning in general.

15 Combination of RepSox with histone deacetylation inhibitors on invitro development competence of porcine somatic cell nuclear transfer embryos

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). In this study, we compared histone deacetylase inhibitors combined with the pluripotency inducer RepSox on invitro development of porcine embryos produced via SCNT. Porcine embryos were treated with valproic acid (VPA), mocetinostat, M344 and panobinostat (LBH589) after SCNT, respectively. The porcine embryo invitro-development competence, histone modification level, and pluripotency-related genes expression were analysed. The results showed that LBH589 significantly increased the blastocyst formation rate compared with mocetinostat, M344, and control. In addition, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2 vs. 67.0%; P<0.05). Furthermore, cotreatment with 12.5mM RepSox and 50 nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9 vs. 8.5%, respectively; P<0.05). Moreover, RepSox + LBH589 improved epigenetic reprogramming by histone acetylation and methylation. The expression of pluripotency-related genes NANOG and SOX2 was found to be significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming, and improves the invitro development of porcine SCNT embryos.

RepSox Increases Porcine Cloning Efficiency by Improving Pluripotency of Donor Nuclei.

Accumulating evidence suggests that a low pluripotency of donor nuclei might lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). To improve the pluripotency of SCNT embryo, RepSox, a defined small-molecule compound that functions in inhibiting the transforming growth factor β signaling pathway, was used to treat nuclear-transferred porcine oocytes after activation. We found that the developmental ability of porcine SCNT embryos (defined as the blastocyst rate) was significantly increased (13.5% vs. 21.8%) when 25 μm RepSox was added to the porcine embryo culturing system for 14-16 hours after activation. Of note, RepSox treatment significantly increased the transcriptional expression of the pluripotency gene NANOG at the four- and eight-cell stages. Furthermore, according to the TUNEL (TdT-mediated dUTP nick end labeling) staining and expression levels of the apoptosis-regulated gene Caspase 3 and proapoptotic gene Bax, the percentage of apoptotic cells in blastocyst cells was not affected after RepSox treatment. These results indicated that treatment with RepSox enhanced the developmental competence of porcine SCNT embryos through improvements in nuclear pluripotency.

Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos.

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P<0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5nM zebularine for 12h compared with the control group (25.4±1.6 vs 11.8±1.7%, P<0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P<0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos.

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5μM RepSox and 50nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P<0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox+LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox+LBH589 group. Moreover, RepSox+LBH589 improved epigenetic reprogramming. In summary, RepSox+LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the invitro development of porcine SCNT embryos.

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

Background/Aims: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. Methods: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. Results: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. Conclusion: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

Nuclear Treatment and Cell Cycle Synchronization for the Purpose of Mammalian and Primate Somatic Cell Nuclear Transfer (SCNT).

Mammalian somatic cell nuclear transfer (SCNT) is a technically and biologically challenging procedure inducing rapid reprogramming of the nucleus from the differentiated into the totipotent state in a few hours. This procedure was initially successfully accomplished in farm animals, then in rodents, and more recently in primates and in humans. Though ethical concerns regarding SCNT still exist, this procedure can be utilized to generate patient and disease-specific pluripotent embryonic stem cell lines, which carry a great promise in improving our understanding of major disease conditions and a hope for better therapies and regenerative medicine. In this section, we will survey the existing literature and describe how mouse SCNT is performed and the importance of donor cell treatment and cycle synchronization prior to SCNT.

Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos.

DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.

Epigenetic reprogramming, gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor--m-carboxycinnamic acid bishydroxamide (CBHA).

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0034-3) contains supplementary material, which is available to authorized users.

48 QUIESCENCE INDUCES LONG-TERM EPIGENETIC CHANGES IN BOVINE FIBROBLASTS THAT IMPROVE THEIR REPROGRAMMING INTO CLONED ANIMALS

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) bovine adult male fibroblasts versus non-starved, diploid G1 controls. Mechanically synchronized G1 cells were generated by manual selection or mitotic shake-off and processed within 3 h post-mitosis. Based on morphological assessment and 5-ethyl-2′-deoxyuridine (EdU) incorporation during continuous labelling, &gt;93% of cells were captured in G1. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay (ELISA), we show that G0 fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3, and H3K27me3, but not H3K9me1. They were also significantly hypoacetylated at H3K9 and H4K5, hyperacetylated at H4K12, and unchanged at H4K16 positions. Furthermore, G0 cells significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as polycomb group proteins EED, SUZ12, PHC1, and RING2. Following NT into metaphase-arrested oocytes, G0 chromatin condensed slower than that of G1 cells, indicating a more relaxed configuration. After 7 days of in vitro culture, H3K9me3, but not H4K4me3, H3K27me3, SUZ12, and RING2, remained hypomethylated in G0- versus G1-derived NT blastocysts, both in the inner cell mass and trophectoderm (730 v. 550 nuclei from 55 v. 42 G0 v. G1 blastocysts, respectively; n = 7 NT runs). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. Expression of other pluripotency-related factors (NANOG, SOX2, STELLA, and IIFITM3), imprinted genes (SNRPN), and histone demethylases (KDM4A) was not affected in G0-derived blastocysts (32 G0 v. 55 G1 blastocysts; n = 4). Following NT, G0 donors developed significantly better into cloned blastocysts (175/382 = 46% v. 122/332 = 37% for G0 v. G1, respectively; n = 7, P &lt; 0.05). Likewise, after transfer into surrogate mothers, G0-derived blastocysts developed significantly better into live calves (5/18 = 28% v. 1/25 = 4% for G0 v. G1, respectively; n = 2, P &lt; 0.05). In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability. This research was supported by FRST C10X0303.

Cell Reprogramming by Cell Extract Treatment*

Cellular reprogramming described a switch of one kind of cell to that of another unrelated cell type by specific methods,which included mammalian somatic cell nuclear transfer,cell fusion,induction of pluripotency by ectopic gene expression,and cell-free extract treatment.Now,more and more researches have proved that cell extract treatment is an important strategy of cellular reprogramming,thus the mechanism and the applicative prospect of this technology are summarized in this review.

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos

Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.

Epigenetic Reprogramming Induced Pluripotency

BACKGROUND: The ability to reprogram mature cells to an embryonic-like state by nuclear transfer or by inducing the expression of key transcription factors has provided us with critical opportunities to linearly map the epigenetic parameters that are essential for attaining pluripotency.CONTENT: Epigenetic reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible tissue, such as skin in the same individual. This has potential applications for cell replacement without the immunosuppression treatments commonly required when cells are transferred between genetically different individuals.SUMMARY: Reprogramming with transcription factors offers tremendous promise for the future development of patient-specific pluripotent cells and for studies of human disease. The identification of optimized protocols for the differentiation of iPS cells and ES cells into multiple functional cell types in vitro and their proper engraftment in vivo will be challenged in the coming years. Given that the first small molecule approaches aimed at activating pluripotency genes have already been devised and that murine iPS cells have recently been derived by using non-integrative transient expression strategies of the reprogramming factors, we expect that human iPS cells without permanent genetic alterations will soon be generated.KEYWORDS: epigenetics, reprogramming, pluripotency, stem cells, iPS cells, chromatin, DNA methylation

Nuclear Reprogramming in Cells

Nuclear reprogramming describes a switch in gene expression of one kind of cell to that of another unrelated cell type. Early studies in frog cloning provided some of the first experimental evidence for reprogramming. Subsequent procedures included mammalian somatic cell nuclear transfer, cell fusion, induction of pluripotency by ectopic gene expression, and direct reprogramming. Through these methods it becomes possible to derive one kind of specialized cell (such as a brain cell) from another, more accessible, tissue (such as skin) in the same individual. This has potential applications for cell replacement without the immunosuppression treatments that are required when cells are transferred between genetically different individuals. This article provides some background to this field, a discussion of mechanisms and efficiency, and comments on prospects for future nuclear reprogramming research.