Donor Nuclei Research Articles

Targeted histone demethylation improves somatic cell reprogramming into cloned blastocysts but not postimplantation bovine concepti†.

Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

Dog cloning from post-mortem tissue frozen without cryoprotectant

Successful reproductive cloning depends on obtaining intact donor nuclei from viable cells, ideally isolated by tissue biopsy of a living donor. However, owners and veterinarians often freeze deceased animals, which eventually causes damage to cellular micro-organelles due to the formation of intracellular water crystals. In the present study, we have reported the production of viable cloned puppies using donor nuclei of cells obtained from frozen carcasses. Five cases of deceased and frozen canine specimens were presented to be cloned. Skin fibroblast cell lines were successfully established for four specimens. Significant longer time was needed for the cell growth from frozen tissues (4 days) to reach 80% confluency compared to fresh tissue and frozen tissues frozen for 1- or 2-days. Similarly, SA-βgal positive cells (death cells) were significantly higher in frozen cells for 2- or 4- days compared to samples from fresh or frozen (1 day) sources. The cloning efficiency (CE) and the pregnancy rates (PR) of frozen cells were lower than those obtained from fresh or living donors (CE 2.4 ± 1.8% vs. 0.6 ± 0.3%, PR 21.7 ± 16.1% vs. 7.7 ± 5.3% for fresh vs. frozen, respectively). Here we demonstrate is the possibility to produce healthy offspring from cell lines obtained from frozen tissue collected post-mortem.

The Effect of Cryopreservation Method to the Quality of Bovine Cumulus Cells Primary Culture

Cumulus cells are play a role as nucleus donor in Somatic Cell Nuclear Transfer (SCNT) technology. This study was conducted to find a suitable method for cryopreserving cumulus cells in aim to provide donor cell stock of primary cell culture. The freezing temperature was made at three levels: -20°C, -80 °C, and -196 °C (liquid nitrogen), while cryopreservation was performed by adding cryoprotectants (CPs) that made from 10% Ethylene Glycol (EG) or 10% Dimethyl Sulfoxide (DMSO) in Dulbecco’s Phosphate Buffer Saline (DPBS) + 20% New Born Calf Serum (NBCS) medium. The results showed that the freezing temperature, cryoprotectants and their interaction had no significant effects on the cell concentration (p>0.05). The cell viability efficiency was decreased significantly when the cell were freezing at -20°C compared to freezing temperature at -80°C and -196°C (p<0.05). The highest cell viability efficiency was obtained from the cell preserved at -196°C using 10% DMSO. The regression test showed that the decrease in temperature will increase the viability efficiency. In conclusion, cumulus cell primary culture can be cryopreserved in temperature of - 80°C and -196°C with CPs of 10% DMSO or 10%EG.

Establishment of porcine nuclear transfer-derived embryonic stem cells using induced pluripotent stem cells as donor nuclei.

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

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&amp;lt;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&amp;lt;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.

A Coumarin-Benzothiazole Derivative as a FRET-Based Chemosensor of Adenosine 5′-Triphosphate

A coumarin-benzothiazole ratiometric probe of ATP was designed and synthesized. The probe is based on incorporation of benzothiazole scaffold as a donor and coumarin nucleus as an acceptor in a single Förster resonance energy transfer/fluorescence resonance energy transfer (FRET) sensing platform. The sensor can detect ATP in aqueous solution with high selectivity over other nucleotide polyphosphate (NPP) anions. Binding of ATP to the sensor results in modulation of FRET efficiency between the donor and the acceptor which afforded a linear relationship between FRET signal and ATP (0.1–10 μM). A limit of detection (LOD) of 94.5 nM was quantified for FRET sensing of ATP by the probe. In addition, Job plot analysis revealed 1:1 binding interaction between the probe and ATP. The FRET probe was successfully utilized in monitoring ATP hydrolysis by apyrase in aqueous solution.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p<.05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p<.05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p<.05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

Do the Cytoplast and Nuclear Material of Germinal Vesicle Oocyte Support Developmental Competence Upon Reconstruction with Embryonic/Somatic Nucleus.

Maturation conditions and oocytes quality have substantial roles on developmental competence of unreconstructed or reconstructed oocytes. Cloning has been reported successfully with low efficiency through embryonic or somatic nuclear transfer into enucleated metaphase II oocytes. It has been suggested that introducing embryonic or somatic nucleus to cytoplast at earlier stage might improve reprogramming of the introduced nucleus. In addition, the synchronization between the donor nucleus and recipient cytoplasts might effect on reprogramming and further embryonic development. Therefore, the question arises; does the cytoplast of germinal vesicle (GV) oocyte containing nuclear sap improve developmental competence upon reconstruction with embryonic/somatic nucleus compared with MII cytoplast. It has been indicated that GV material is essential for remodeling of sperm or embryonic or somatic nucleus in GV oocyte cytoplast and their further embryonic developmental competence. GV cytoplasts could be obtained through micromanipulation. Different micromanipulation techniques of immature oocytes at different stages were adapted in addition to introducing donor nuclei at G0/G1, S and G2/M phase, and enucleolation technique as well. Upon micromanipulation, it could obtain GV cytoplasts; cumulus-free and without GV material, cumulus-complexes and without GV material, cumulus-free and with GV material, and cumulus-complexes and with GV material in addition to enucleolated GV oocytes. Therefore, this short review will address briefly the importance of maturation conditions, cumulus cells, oocyte quality, the techniques of enucleation GV oocyte, the cell cycle stage of the introduced donor cell, or nucleus for oocyte maturation and embryo development.

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.

Cloning in livestock agriculture

A tremendous amount is required of the oocyte cytoplasm to reprogramme a differentiated donor nucleus after somatic cell nuclear transfer so that it re-acquires a state of totipotency and can form a cloned individual. These reprogramming events must occur in a relatively short period after embryo reconstruction, quite unlike the situation during gametogenesis. It is remarkable that nuclear transfer can produce physiologically normal animals, but the process is highly prone to epigenetic errors. Aberrant patterns of gene expression are believed to contribute to the cumulative losses and abnormal phenotypes observed throughout development, from embryonic to adult stages. As a consequence, cloning efficiencies with current nuclear transfer technology are low and limit practical applications in farming. These applications depend upon the reliability with which nuclear transfer can reproduce specific genotypes and phenotypes in resulting offspring. Present animal welfare issues are a significant barrier for acceptance of nuclear transfer in agriculture. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. The safety of food products derived from clones also needs to be thoroughly evaluated. The current indication that the sexually derived offspring of nuclear transfer clones are normal provides confidence for the first applications of the technology in agriculture, namely the generation of cloned, genetically elite animals for subsequent breeding. Future improvements in cloning will come from both technical advances and a greater fundamental understanding of the molecular mechanisms of reprogramming with an aim to control this process better. Increased efficiencies should improve the acceptability and utility of cloning technology.

NMR study of optically hyperpolarized phosphorus donor nuclei in silicon

We use above-bandgap optical excitation, via a 1047 nm laser, to hyperpolarize the $^{31}$P spins in low-doped (N$_D =6\times10^{15}$ cm$^{-3}$) natural abundance silicon at 4.2 K and 6.7 T, and inductively detect the resulting NMR signal. The $30$ kHz spectral linewidth observed is dramatically larger than the 600 Hz linewidth observed from a $^{28}$Si-enriched silicon crystal. We show that the observed broadening is consistent with previous ENDOR results showing discrete isotope mass effect contributions to the donor hyperfine coupling. A secondary source of broadening is likely due to variations in the local strain, induced by the random distribution of different isotopes in natural silicon. The nuclear spin T$_1$ and the build-up time for the optically-induced $^{31}$P hyperpolarization in the natural abundance silicon sample were observed to be $178\pm47$ s and $69\pm6$ s respectively, significantly shorter than the values previously measured in $^{28}$Si-enriched samples under the same conditions. We also measured the T$_1$ and hyperpolarization build-up time for the $^{31}$P signal in natural abundance silicon at 9.4 T to be $54\pm31$ s and $13\pm2$ s respectively. The shorter build-up and nuclear spin T$_1$ times at high field are likely due to the shorter electron-spin T$_1$, which drives nuclear spin relaxation via non-secular hyperfine interactions. At 6.7 T, the phosphorus nuclear spin T$_{2}$ was measured to be $16.7\pm1.6$ ms at 4.2 K, a factor of 4 shorter than in $^{28}$Si-enriched crystals. This was observed to further shorten to $1.9\pm0.4$ ms in the presence of the infra-red laser.

Strategies for improvement of cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a powerful tool that is being applied in a variety of fields as diverse as the cloning and production of transgenic animals, rescue of endangered species and regenerative medicine. However, cloning efficiency is still very low and SCNT embryos generally show poor developmental competency and many abnormalities. The low efficiency is probably due to incomplete reprogramming of the donor nucleus and most of the developmental problems are thought to be caused by epigenetic defects. Applications of SCNT will, therefore, depend on improvements in the efficiency of production of healthy clones. This review has summarised the progress and strategies that have been used to make improvements in various animal species, especially over the period 2010–2017, including strategies based on histone modification, embryo aggregation and mitochondrial function. There has been considerable investiagation into the mechanisms that underpin each strategy, helping us better understand the nature of genomic reprogramming and nucleus–cytoplasm interactions.

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos.

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5-methylcytosine-5mC and 5-hydroxymethylcytosine-5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT-♀ and SCNT-♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT-♀ embryos was greater than that of SCNT-♂ embryos (p<0.05). 5mC was mainly expressed in SCNT-♀ embryos, whereas 5hmC was majorly expressed in SCNT-♂ embryos (p<0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT-♂ embryos were higher than those of SCNT-♀ embryos (p<0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight-stage of the IVF, SCNT-♀ and SCNT-♂embryos (p<0.05). However, H3K9me3 was upregulated in SCNT-♂ embryos at the eight-cell stage (p<0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two-cell, eight-cell and blastocysts of SCNT-♂ embryos (p<0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT-♀ embryos than that of SCNT-♂ embryos.

Dynamic changes in chromatin and microtubules at the first cell cycle in SCNT or IVF goat embryos.

We investigated the dynamic changes in chromatin and microtubules at the first cell cycle in goat somatic cell nuclear transfer (SCNT)-derived and in vitro fertilization (IVF)-derived embryos. Stage-dependent and characteristic changes to chromatin and microtubules occurred in SCNT-derived embryos at different times after activation. About half donor nuclei underwent premature chromosome condensation (PCC) at 1 h post activation, and furtherly reached telophase at 2 h after activation. However, we discovered that the separated chromosomes reaggregated, not keeping two independent nuclei; and formed one pronucleus at 2.5 h after activation. One pronucleus was found in all reconstructed oocytes except other no nucleus oocytes from 3 to 22 h after activation. Reconstructed oocytes reached the first mitotic metaphase at 23 h post activation, which was later than that of IVF-derived embryos at 16 h after insemination. SCNT-derived embryos showed significantly higher abnormalities in the first mitotic metaphase spindle, compared with IVF-derived embryos. Abnormal spindles included multi polar and half spindles. SCNT-derived embryos began to cleave at 24 h after activation, which was later than that of IVF-derived embryos at 21 h after insemination. SCNT-derived embryos showed delayed conversion from telophase to interphase than IVF-derived embryos during cleavage. These might lead to poor development in SCNT-derived embryos.

Can Reprogramming of Overall Epigenetic Memory and Specific Parental Genomic Imprinting Memory within Donor Cell-Inherited Nuclear Genome be a Major Hindrance for the Somatic Cell Cloning of Mammals? – A Review

Abstract Successful cloning of animals by somatic cell nuclear transfer (SCNT) requires epigenetic transcriptional reprogramming of the differentiated state of the donor cell nucleus to a totipotent embryonic ground state. It means that the donor nuclei must cease its own program of gene expression and restore a particular program of the embryonic genome expression regulation that is necessary for normal development. Transcriptional activity of somatic cell-derived nuclear genome during embryo pre- and postimplantation development as well as foetogenesis is correlated with the frequencies for spatial remodeling of chromatin architecture and reprogramming of cellular epigenetic memory. This former and this latter process include such covalent modifications as demethylation/re-methylation of DNA cytosine residues and acetylation/deacetylation as well as demethylation/re-methylation of lysine residues of nucleosomal core-derived histones H3 and H4. The main cause of low SCNT efficiency in mammals turns out to be an incomplete reprogramming of transcriptional activity for donor cell-descended genes. It has been ascertained that somatic cell nuclei should undergo the wide DNA cytosine residue demethylation changes throughout the early development of cloned embryos to reset their own overall epigenetic and parental genomic imprinting memories that have been established by re-methylation of the nuclear donor cell-inherited genome during specific pathways of somatic and germ cell lineage differentiation. A more extensive understanding of the molecular mechanisms and recognition of determinants for epigenetic transcriptional reprogrammability of somatic cell nuclear genome will be helpful to solve the problems resulting from unsatisfactory SCNT effectiveness and open new possibilities for common application of this technology in transgenic research focused on human biomedicine.

Additive Expression of hTBM on hCD46 Stable Cell Line Has Synergistic Effect on Complement Regulation

Background Coagulation dysregulation and intravascular thrombosis during acute humoral xenograft rejection (AHXR) still remains the major obstacle in solid organ xenotransplantation. TBM or CD46 have been reported as an anti-coagulant or complement regulatory molecules and also potent anti-inflammatory molecules. In this study, we showed whether additional TBM expression on CD46 stable cell has synergistic effects on anti-coagulant or complement regulation. Methods & Results Fetal pig fibroblasts were transfected with hTBM-hCD46 vector and colonies expressing high amounts of the fusion protein were selected and used as nucleus donor for somatic cell nuclear transfer (SCNT) and three gilts became pregnant and one farrowed one live male piglet, which was no healthy and moved to the SPF facility but died one day after and two of the pregnant gilts, however, aborted during gestation. We isolated fibroblast from the ear of TG pig expressing hTBM-hCD46 and check the expression levels of each genes and the regulatory functions on complement and coagulation. Serum toxicity was significantly reduced in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast at 10% or 20% serum treatment. Thrombin generation was dramatically reduced and coagulation time was prolonged in the fibroblast from hTBM-hCD46 TG pig compared with wild type pig’s fibroblast. Conclusion The fibroblast from hTBM-hCD46 TG pig has synergistic effects on anti-coagulant or complement regulation therefore, genetically modifying pigs expressing TBM and CD46 would be a promising candidate for xenotransplantation to prolong the graft survival.

Influence of storage time and nutrient medium on recovery of fibroblast-like cells from refrigerated collared peccary (Pecari tajacu Linnaeus, 1758) skin.

Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4-6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4-6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50d did not yield any somatic cells. Cells recovered from explants stored for 10d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50d in the presence of medium, and tissues refrigerated for up to 10d in the presence of medium yielded more viable cells.

Cytoplast source influences development of somatic cell nuclear transfer (SCNT) embryos in vitro but not their development to term after transfer to synchronized recipients in dromedary camels (Camelus dromedarius)

Studies were conducted to evaluate the adequate time for exposure of donor nucleus to oocyte cytoplast before its activation and the effect of oocyte source on the development of SCNT embryos in camels. A higher number of embryos cleaved and developed to blastocyst stage (P < 0.05) when couplets were activated between 1 and 2 h-than that of those activated at 0.5 h or more than 2 h post-fusion. A reduced number of reconstructed embryos cleaved (55.2 ± 7.6%) and developed to the blastocyst stage (20.5 ± 5.5%) when in vitro matured oocytes collected from the slaughterhouse were used as donor cytoplasts, compared to in vitro (71.3 ± 1.3 and 36.7 ± 7.3%) or in vivo matured (91.7 ± 8.3 and 35.4 ± 6.0%) oocytes obtained from live animals (P < 0.05), respectively. However, no differences were observed between the different types of oocyte sources on the establishment of pregnancies and delivery of offspring's. In conclusion, couplets activated 1–2 h post-fusion had higher in vitro developmental potential and oocytes collected from live animals were better in supporting the cleavage and blastocyst production in vitro than oocytes collected from slaughterhouse ovaries, however, all sources of oocytes can be utilized as donor cytoplasts and have the potential to support development of full-term calves after transfer into synchronized recipients.

Cloning of Macaque Monkeys by Somatic Cell Nuclear Transfer

Generation of genetically uniform non-human primates may help to establish animal models for primate biology and biomedical research. In this study, we have successfully cloned cynomolgus monkeys (Macaca fascicularis) by somatic cell nuclear transfer (SCNT). We found that injection of H3K9me3 demethylase Kdm4d mRNA and treatment with histone deacetylase inhibitor trichostatin A at one-cell stage following SCNT greatly improved blastocyst development and pregnancy rate of transplanted SCNT embryos in surrogate monkeys. For SCNT using fetal monkey fibroblasts, 6 pregnancies were confirmed in 21 surrogates and yielded 2healthy babies. For SCNT using adult monkey cumulus cells, 22 pregnancies were confirmed in 42surrogates and yielded 2 babies that were short-lived. In both cases, genetic analyses confirmed that the nuclear DNA and mitochondria DNA of the monkey offspring originated from the nucleus donor cell and the oocyte donor monkey, respectively. Thus, cloning macaque monkeys by SCNT is feasible using fetal fibroblasts.

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.