Cloning Efficiency Research Articles

Porcine somatic cell nuclear transfer using telomerase reverse transcriptase-transfected mesenchymal stem cells reduces apoptosis induced by replicative senescence

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

Effects of variation in the number and developmental stage of donor embryos and ovulation status of the surrogate mother on the efficiency of pig somatic cell cloning

Effects of variation in the number and developmental stage of donor embryos and ovulation status of the surrogate mother on the efficiency of pig somatic cell cloning

Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs) Using Bacterial Whole Cells as Molecular Probes.

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation.

There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.

TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

Pregnancy and Calving Rates of Cloned Dromedary Camels Produced by Conventional and Handmade Cloning Techniques and In Vitro and In Vivo Matured Oocytes.

Despite practical implication of cloning in camelids, its broad application has been hampered by technical and biological problems. Method of somatic cell nuclear transfer (SCNT) and oocyte competence are the principal technical and biological factors, respectively, that determine the cloning efficiency. This study, therefore, investigated differential contributions of two SCNT methods [modified handmade cloning (mHMC) vs. conventional (cNT)] and two recipient oocyte sources [abattoir-derived (Vitro) vs. FSH-stimulated (Vivo)] on the efficiency of dromedary camel cloning. The mHMC method supported similar rates of fusion, cleavage, and total blastocyst development, compared to conventional NT (cNT) (94, 89.1, and 68.5% vs. 78.9, 92, and 73.5%, respectively) when Vivo oocytes are used. However, using Vitro oocytes, mHMC supported significantly higher rates for these criteria, except for the cleavage, compared to cNT (95.5, 76.2, 25.2% vs. 75.3, 76.7, and 13.9%, respectively). A total of seven clones were born from mHMC/Vitro (four calves), mHMC/Vivo (one calf), cNT/Vitro (one calf), and mHMC/Vivo&Vivo (one calf)-derived embryos with overall efficiencies of 31.9, 26.6, 20, and 30% for initial pregnancy, 10.6, 6.6, 7.5, and 5% for development to term, and 8.5, 6.6, 2.5, 5% for development to weaving, respectively. To conclude, the quality of recipient oocyte greatly impacts cloning efficiency in vitro with no apparent carrying over effect on cloning efficiency in vivo, but the efficiency of SCNT method may compensate for the initial poor oocyte competence during in vitro and in vivo development of cloned embryos. The introduced mHMC could be a superior alternative to conventional method for simple, fast, and efficient production of cloned offspring in camelids.

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.

Optimizing 5-aza-2′-deoxycytidine treatment to enhance the development of porcine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming

Apoptosis and incomplete DNA methylation reprogramming in cloned embryos reduce cloning efficiency. 5-aza-2′-deoxycytidine (5-aza-dC) is proven to regulate apoptosis and DNA methylation reprogramming, however, the treatment method and potential role of 5-aza-dC during cloned embryo development are still not well studied. This study displayed that treating donor cells with 5-aza-dC (AN group) significantly reduced the blastocyst rate, while treating cloned embryos (NA group) or both donor cells and cloned embryos (ANA group) significantly promoted the blastocyst formation, and the ANA group was the best treatment of 5-aza-dC to enhance the development of cloned embryos. Then, compared with the NT group, the ANA group showed the significantly enhanced nuclear remodeling. The apoptotic cell numbers and rates of blastocysts were significantly reduced, and the expression levels of significantly upregulated anti-apoptosis gene Bcl2l1 and downregulated pro-apoptosis genes Bax, P53 and Caspase3 were observed in the ANA group. Further study demonstrated that the transcription levels of DNA methylation reprogramming genes Dnmt1, Dnmt3a, Tet1 and Tet3 were significantly upregulated, and, significant genomic DNA remethylation, DNA demethylation of pluripotency gene Oct4, and DNA remethylation of tissue specific gene Thy1 were observed at the blastocyst stage in the ANA group. Embryo development related genes including Igf2, H19, Oct4, Nanog, Sox2, Eif1a, Cdx2 and ATP1b1 were significantly upregulated, and Thy1 and Col5a2 were remarkably silenced at the 4-cell and blastocyst stages in the ANA group. In conclusion, the best 5-aza-dC treatment enhanced the development of cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming.

Overcoming Intrinsic H3K27me3 Imprinting Barriers Improves Post-implantation Development after Somatic Cell Nuclear Transfer.

Successful cloning by somatic cell nuclear transfer (SCNT) requires overcoming significant epigenetic barriers. Genomic imprinting is not generally regarded as such a barrier, although H3K27me3-dependent imprinting is differentially distributed in E6.5 epiblast and extraembryonic tissues. Here we report significant enhancement of SCNT efficiency by deriving somatic donor cells carrying simultaneous monoallelic deletion of four H3K27me3-imprinted genes from haploid mouse embryonic stem cells. Quadruple monoallelic deletion of Sfmbt2, Jade1, Gab1, and Smoc1 normalized H3K27me3-imprinted expression patterns and increased fibroblast cloning efficiency to 14% compared with a 0% birth rate from wild-type fibroblasts while preventing the placental and body overgrowth defects frequently observed in cloned animals. Sfmbt2 deletion was the most effective of the four individual gene deletions in improving SCNT. These results show that lack of H3K27me3 imprinting in somatic cells is an epigenetic barrier that impedes post-implantation development of SCNT embryos and can be overcome by monoallelic imprinting gene deletions in donor cells.

Maternal H3K27me3-dependent autosomal and X chromosome imprinting.

Genomic imprinting and X-chromosome inactivation (XCI) are classic epigenetic phenomena that involve transcriptional silencing of one parental allele. Germline-derived differential DNA methylation is the best-studied epigenetic mark that initiates imprinting, but evidence indicates that other mechanisms exist. Recent studies have revealed that maternal trimethylation of H3 on lysine 27 (H3K27me3) mediates autosomal maternal allele-specific genesilencing and has an important role in imprinted XCI through repression of maternal Xist. Furthermore, loss of H3K27me3-mediated imprinting contributes to the developmental defects observed in cloned embryos. This novel maternal H3K27me3-mediated non-canonical imprinting mechanism further emphasizes the important role of parental chromatin in development and could provide the basis for improving the efficiency of embryo cloning.

SiRNA inhibition and not chemical inhibition of Suv39h1/2 enhances pre-implantation embryonic development of bovine somatic cell nuclear transfer embryos.

The efficiency of somatic cell nuclear transfer (SCNT) is low due to the strong resistance of somatic donor cells to epigenetic reprogramming. Many epigenetic drugs targeting DNA methylation and histone acetylation have been used in attempts to improve the in vitro and in vivo development of SCNT embryos. H3K9me3 has been shown to be an important reprogramming barrier for generating induced pluripotent stem cells (iPSCs) and SCNT embryos in mice and humans. In this study, we examined the effects of selective siRNA and chemical inhibition of H3K9me3 in somatic donor cells on the in vitro development of bovine SCNT embryos. Chaetocin, an inhibitor of SUV39H1/H2, was supplemented during the culture of donor cells. In addition, the siRNA knockdown of SUV39H1/H2 was performed in the donor cells. The effects of chaetocin and siSUV39H1/H2 on H3K9me3 and H3K9ac were quantified using flow cytometry. Furthermore, we assessed chaetocin treatment and SUV39H1/H2 knockdown on the blastocyst formation rate. Both chaetocin and siSUV39H1/H2 significantly reduced and elevated the relative intensity level of H3K9me3 and H3K9ac in treated fibroblast cells, respectively. siSUV39H1/H2 transfection, but not chaetocin treatment, improved the in vitro development of SCNT embryos. Moreover, siSUV39H1/H2 altered the expression profile of the selected genes in the derived blastocysts, similar to those derived from in vitro fertilization (IVF). In conclusion, our results demonstrated H3K9me3 as an epigenetic barrier in the reprogramming process mediated by SCNT in bovine species, a finding which supports the role of H3K9me3 as a reprogramming barrier in mammalian species. Our findings provide a promising approach for improving the efficiency of mammalian cloning for agricultural and biomedical purposes.

The in vitro development of cloned sheep embryos treated with Scriptaid and Trichostatin (A).

Introduction and aimsAlthough, it has been success in the generation of animal clones from somatic cells in various animal species, the information related to nuclear reprogramming of cloned embryos is found to be limited. This study aims to compares the effect of both Scriptaid (SCR) and Trichostatin (A) treatments in improving cloning efficiency, and embryos developmental rate of cloned sheep embryos in vitro. Three groups were formed, i.e., one SCR group, second TSA group, with both treatment concentrations of 5 nM, 50 nM, and 500 nM, respectively, and third were control group with 0 nM. Methods: Ovaries of slaughtered sheep were collected and oocytes were recovered from antral follicles using aspiration method and in vitro maturation of oocytes were done. Then zona dissecting with micropipettes and oocyte enucleation were carried out under the micromanipulator. Later nuclear transfer, cell fusion and activation were done via cell fusion machine. Finally the embryo cultured in incubating chamber at the CO2 incubator up to 9 days. The result: In general the results showed that when the concentration increases the cleavage rate increased. The cleavage rates of the SCNT embryos treated with SCR at different concentrations are closely related to cleavage rate of embryos treated with TSA at same concentration; such as 39.47% for 500 nM TSA, 38.09% for 500 nM SCR; 18.6% for 50 nM TSA, 19.17% for 50 nM SCR, and 22.64% for 5 nM TSA, 17.18% for 5 nM SCR. As for the control group, the cleavage rate of the SCNT embryos cleavage ratewere27.47%., 30% and 30.85% respectively for bothtreatments. While there is a significant difference in TSA treatments at an eight-cell stage at the concentration (5 and 50 nM TSA) compared to the all other cleavage cell stages of (500 nM TSA and control). Also their were a differences between (50 nM of TSA) compared to the (50 nM SCR). Also there were a significant differences between the 16 cell stage at the (500 nM TSA) compared to other treatment (5 nM, 50 nM TSA and control). Regarding the SCR there were a significant difference at 8 cell stage between (5 nM SCR), compared to the other treatment (50 nM, 500 nM SCR and control). Also there were a significant difference at 16 cell stage between (50 nM, and 500 nM SCR), compared to the other treatment (5 nM SCR and control). While in the development of the embryos reach to blastocyst stage the SCR and the control group show a higher rate, in compered to TSA that did not show any development to blastocyst stage. The total SCR treatment showed (3/41 = 7.31%), and the total control showed (4/89 = 4.49%) blastula stage. It concludes that SCR improve the final development blastula stage compared to the TSA treatments that did not improved embryos reach to final developmental blastula stages may be due to spices differences or to the toxicity of TSA, especially at higher concentrations.

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.

Fusion-PCR generates attL recombination site adaptors and allows Rapid One-Step Gateway (ROG) cloning

Gateway recombination-based cloning, which eliminates the use of restriction endonucleases and ligase, has been widely used for the construction of high-throughput (HTP) vectors. However, this approach is very expensive and its two-stage reaction process is laborious and time consuming. Therefore, we developed a Gateway cloning method that uses fusion-PCR to generate attL recombination site adaptors, and the PCR products, which can be directly cloned into destination vectors, giving rise to Rapid One-Step Gateway (ROG) Cloning. 100% of cloning efficiencies were obtained by this ROG method. This method has no BP reaction/entry clone step, thus halving the cost and time consumed. Overall, this work provides a highly efficient, rapid, low-cost method for directional recombination cloning.

Influence of stand density on growth and water use efficiency in Eucalyptus clones

We examined the influence of stand density and genotype on transpiration and water use efficiency in high productivity plantations. Three widely planted Eucalyptus clones that differ in drought tolerance and productivity (E. urophylla, E. urophylla × E. grandis and E. grandis × E. camaldulensis, clones IP, B2 and C3, respectively) were measured at four densities (590, 1030, 1420, and 2950 trees ha−1). Over the 1-year study period (1.5–2.5 years after planting), individual biomass increment decreased with increasing density, from 21 kg tree−1 at 590 trees ha−1 to 6 kg tree−1 at 2950 trees ha−1. Stand increment typically follows the reverse pattern, increasing as density increases. This was the case for two clones (IP and B2), but stand increment was consistent across tree spacings for C3. Transpiration increased with density, from a low of 622 mm yr−1 to a high of 879 mm y−1. Some of the increased water use resulted from higher leaf area index at higher densities. The B2 clone transpired the most water on average, produced the greatest increment (23 Mg ha−1 yr−1 for 1030 trees ha−1), and produced the most wood L−1 transpiration (water use efficiency, 2.3 g biomass L−1). The clone C3 had the lowest increment (only 12 Mg ha−1 yr−1) because of the combination of low transpiration and low water use efficiency (only 1.5 g biomass L−1). Optimizing clone selection and silviculture for the combination of high yield and high water use efficiency may help reduce risks from drought as well as water conservation.

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.

Melatonin Enhances the Development of Porcine Cloned Embryos by Improving DNA Methylation Reprogramming.

Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Melatonin has been proven to improve the development of cloned embryos, however, the role of melatonin during somatic cell nuclear transfer remains unclear. This work demonstrated that 10-7 M melatonin significantly enhanced the developmental progress, reduced the arrested rate before zygotic genome activation, and upregulated the blastocyst rate of cloned embryos. Melatonin also promoted the pseudo-pronucleus formation, increased blastocyst cell number, and reduced embryo apoptosis through upregulating the expression of antiapoptosis factors while downregulating the transcription of proapoptosis genes. Further study displayed that DNA methylation reprogramming related genes were greatly improved in cloned embryos when treated with melatonin; then, melatonin effectively promoted genomic DNA demethylation and DNA remethylation, DNA demethylation of pluripotency related gene Oct4, DNA methylation maintenance of imprinted gene H19/Igf2, and DNA remethylation of tissue-specific gene Thy1 in cloned embryos. Thus, zygotic genome activation related gene Eif1a, pluripotency related genes Oct4, Nanog, and Sox2, imprinted genes Igf2 and H19, and blastocyst quality related genes Cdx2 and ATP1b1 were remarkably upregulated, and tissue-specific genes Thy1 and Col5a2 were considerably silenced. In conclusion, melatonin enhanced the development of cloned embryos by ameliorating DNA methylation reprogramming. This work reveals that melatonin can regulate DNA methylation reprogramming and provides a novel insight to improve cloning efficiency.

Epigenetic Reprogramming During Somatic Cell Nuclear Transfer: Recent Progress and Future Directions.

Somatic cell nuclear transfer (SCNT) has broad applications but is limited by low cloning efficiency. In this review, we mainly focus on SCNT-mediated epigenetic reprogramming in livestock and also describe mice data for reference. This review presents the factors contributing to low cloning efficiency, demonstrates that incomplete epigenetic reprogramming leads to the low developmental potential of cloned embryos, and further describes the regulation of epigenetic reprogramming by long non-coding RNAs, which is a new research perspective in the field of SCNT-mediated epigenetic reprogramming. In conclusion, this review provides new insights into the epigenetic regulatory mechanism during SCNT-mediated nuclear reprogramming, which could have great implications for improving cloning efficiency.

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

RNA sequencing revealed the abnormal transcriptional profile in cloned bovine embryos

Somatic cell nuclear transfer (SCNT) has potential applications in agriculture and biomedicine, but the efficiency of cloning is still low. In this study, the transcriptional profiles in cloned and fertilized embryos were measured and compared by RNA sequencing. The 2-cell embryos were detected to identify the earliest transcriptional differences between embryos derived through IVF and SCNT. As a result, 364 genes showed decreased expression in cloned 2-cell embryos and were enriched in “intracellular protein transport” and “ubiquitin mediated proteolysis”. In blastocysts, 593 genes showed decreased expression in cloned blastocysts and were enriched in “RNA binding”, “nucleotide binding”, “embryo development”, and “adherens junction”. We identified 14 development related genes that were not activated in the cloned embryos. Then, 68 and 245 long non-coding RNAs were recognized abnormally expressed in cloned 2-cell embryos and cloned blastocysts, respectively. Furthermore, we found that incomplete RNA-editing occurred in cloned embryos and might be caused by decreased ADAR expression. In conclusion, our study revealed the abnormal transcripts and deficient RNA-editing sites in cloned embryos and provided new data for further mechanistic studies of somatic nuclear reprogramming.