Somatic Cell Nuclear Transfer Research Articles

Transcriptional profiling of oocyte maturation and embryonic development elucidates metabolism and control of development

With the advent of next generation sequencing platforms (RNA-seq), transcriptional profiling permits the characterization of millions of RNAs from even the most limiting samples like early embryos. High-throughput RNA-seq can generate over 600 gigabases (Gb) in a single sequencing run, providing a near-complete record of all of the genes expressed in a sample at the time of collection. Condensing and finding coherence in the immense amount of raw data generated by transcriptional profiling methods such as RNA-seq is a complex task, but necessary if useful information is to be gleaned. Here we review the current technology and describe how transcriptional profiling has been used to improve oocyte maturation and embryo culture conditions, to decrease polyspermy and to improve somatic cell nuclear transfer. Most recently, RNA-seq data has provided a unique framework for understanding metabolism of the early embryo, i.e. the Warburg Effect. Rapidly proliferating cells use glucose for synthesis of nucleotides that are necessary for DNA synthesis. They shunt metabolism away from the tri-carboxylic acid cycle and toward lactic acid production and the pentose phosphate pathway. Pathways identified by RNA-seq data show that early embryos, as it turns out, are quite similar and thrive in conditions that promote proliferation of cancer cells. Application of the Warburg Effect framework to early embryos has, and will continue to contribute to improved culture conditions for embryos in vitro.

Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN−/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.

Determining Influence of Culture Media and Dose-Dependent Supplementation with Basic Fibroblast Growth Factor on the Ex Vivo Proliferative Activity of Domestic Cat Dermal Fibroblasts in Terms of Their Suitability for Cell Banking and Somatic Cell Cloning of Felids

Abstract Dermal fibroblasts are commonly used as donors of genetic material for somatic cell nuclear transfer in mammals. Basic fibroblast growth factor (bFGF) is a cytokine that regulates proliferation and differentiation of different cell types. The study was aimed at optimizing the cell culture protocol for cat dermal fibroblasts by assessing the influence of culture media and different doses of bFGF on proliferation of fibroblasts and their viability in terms of cell banking and somatic cloning of felids. In Experiment I, skin biopsies of domestic cats were cultured in DMEM (D) and/or DMEM/F12 (F), both supplemented with 5 ng bFGF/ml (D-5, F-5, respectively). After the primary culture reached ~80% of confluency, the cells were passaged (3–4 times) and cultured in media with (D-5, F-5) or without (D-0, F-0) bFGF. To determine the optimal doses of bFGF, in Experiment II, secondary fibroblasts were cultured in DMEM with 0 (D-0), 2.5 (D-2.5), 5 (D-5) or 10 (D-10) ng bFGF/ml. The results showed that in D-5 the cells proliferated faster than in D-0, F-5 and F-0. Due to their poor proliferation, passages IV were not performed for cells cultured in F-0. In experiment II, a dose-dependent effect of bFGF on proliferation of cat dermal fibroblasts was found. In D-5 and D-10, the cells exhibited higher (P<0.05) proliferation compared with D-0. In D-2.5 the cells showed a tendency to proliferate slower than in D-5 and D-10 and at the same faster than in D-0. In conclusion. DMEM supplemented with bFGF provides better proliferation of domestic cat dermal fibroblasts culture than DMEM/F12. Supplementation of culture medium with bFGF has a beneficial effect on cat dermal fibroblast proliferation and could be recommended for addition to culture media.

Gene Editing of Swine Myosin Vb Induces Microvillus Inclusion Disease and Loss of Apical Sodium Transporters with Maintenance of CFTR in Enterocytes

BackgroundThe human genetic disorder Microvillus Inclusion Disease (MVID) results from inactivating mutations in Myosin Vb (MYO5B). MVID is characterized by severe, unremitting diarrhea that typically presents shortly after birth. The necessity of implementing total parenteral nutrition (TPN) and the long‐term administration of TPN in neonates with MVID presents challenges to elucidating the complex pathogenesis of MVID in humans. Thus, a large animal model of MVID that closely recapitulates human physiology is needed. To better understand the pathophysiology of MVID we created a large animal model that closely mimics human MVID.MethodsPigs were cloned using somatic cell nuclear transfer of primary pig fibroblasts edited with TALEN to express a P663L mutation of the MYO5B gene. This swine P663L mutation, referred to as MYO5B(P663L), corresponds to the Navajo P660L mutation seen in MVID patients. Neonatal pigs were euthanized within 1 day of birth and tissue was collected for analysis and generation of intestinal enteroids.ResultsThe MYO5B(P663L) pigs developed diarrhea shortly after birth, similar to patients with MVID. Immunostaining for brush border components F‐actin, ezrin, CD10 and DPPIV showed the presence of subapical intracellular inclusions in enterocytes of MYO5B P663L pigs. Transmission electron microscopy confirmed the presence of inclusions lined with microvilli in the MYO5B(P663L) pigs. Immunostaining for NHE3 and SGLT1, drivers of intestinal sodium and water absorption, showed decreased apical expression and diffuse sub‐apical expression in the MYO5B(P663L) pigs compared to control pigs. In contrast to decreased expression of sodium transporters, CFTR was maintained on the apical membrane of MYO5B(P663L) pigs. Enteroids generated from the proximal small intestine of neonatal control and MYO5B(P663L) pigs grew at similar rates, but showed differences in the brush border. Enteroids derived from MYO5B(P663L) pigs had sub‐apical expression of intestinal alkaline phosphatase and DPPIV and decreased apical expression of NHE3 compared to control enteroids. Administration of forskolin resulted in increased swelling in MYO5B(P663L) enteroids (170.8% ± 15.9) compared to control enteroids (90.4% ± 4.6), indicative of functional Cl− secretion.ConclusionsCollectively, these data suggest that decreased apical expression of NHE3 and SGLT1, which are required for water absorption, are likely responsible for the MVID diarrhea disorder. Moreover, maintenance of functional CFTR on the apical membrane of enterocytes in the MYO5B(P663L) pig may further exacerbate the development of diarrhea by actively secreting Cl−, thus driving further water loss by facilitating fluid secretion. This novel large animal model of MVID could provide an important vehicle for testing novel therapeutic approaches for patients with MVID.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Production of transgenic cattle expressing lysine-rich polypeptide in milk by somatic cell nuclear transfer.

This study aims to produce transgenic cattle expressing lysine-rich polypeptide in milk by somatic cell nuclear transfer. Lysine is the first limiting amino acid in cereal crops. Lysine deficiency leads to fatigue, nausea and reproductive disorders. In this study, transgenic cells with lysine-rich cDNA (β-casein, αS2-casein and lactotransferrin cDNA) were used as donor cells to produce transgenic cattle by somatic cell nuclear transfer. The transgene integration and expression in transgenic cattle were examined using western blotting, Southern blotting, PCR and RT-PCR. Our results showed that the lysine-rich gene was successfully integrated into the bovine genome and expressed in the mammary gland of transgenic cattle during lactation. The milk lysine concentrations of three transgenic cattle were higher than those of non-transgenic controls. The production of transgenic cattle expressing lysine-rich polypeptides in milk may provide a large-scale and cost-effective resource for lysine-rich products.

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.

Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer.

Mammalian telomere lengths are primarily regulated by telomerase, consisting of a reverse transcriptase protein (TERT) and an RNA subunit (TERC). We previously reported the generation of mouse Terc+/− and Terc−/− embryonic stem cells (ntESCs) by somatic cell nuclear transfer. In the present work, we investigated the germ layer development competence of Terc−/−, Terc+/− and wild-type (Terc+/+) ntESCs. The telomere lengths are longest in wild-type but shortest in Terc−/− ntESCs, and correlate reversely with the population doubling time. Interestingly, while in vitro embryoid body (EB) differentiation assay reveals EB size difference among ntESCs of different genotypes, the more stringent in vivo teratoma assay demonstrates that Terc−/− ntESCs are severely defective in differentiating into the mesodermal lineage cartilage. Consistently, in a directed in vitro chondrocyte differentiation assay, the Terc−/− cells failed in forming Collagen II expressing cells. These findings underscore the significance in maintaining proper telomere lengths in stem cells and their derivatives for regenerative medicine.

The desirable donor pig to eliminate all xenoreactive antigens.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF).

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

Effects of Endoplasmic Reticulum Stress Inhibitor Treatment during the Micromanipulation of Somatic Cell Nuclear Transfer in Porcine Oocytes.

We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; 100 μM) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 (Xbp1), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment (p<0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated Xprotein (Bax) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments (p<0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number (63.0±7.2) were significantly higher (p<0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and 41.7±3.1, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.

Xist Intron 1 Repression by Transcriptional-Activator-Like Effectors Designer Transcriptional Factor Improves Somatic Cell Reprogramming in Mice.

Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming. Stem Cells 2019;37:599-608.

Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

SummaryCryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT) mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopreserved oocyte cytoplasm (SCNT-CROC) was inferior to those using fresh cytoplasm (SCNT-FOC). Using RNA-sequencing analysis, we found upregulation of eight pro-apoptotic-related genes (Cyct, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, and Pmaip) in the SCNT-CROC group. Furthermore, the addition of melatonin, an agent that reduces apoptosis and ROS production, enhanced blastocyst formation rates in the SCNT-CROP group when compared with the melatonin-untreated group. Additionally, melatonin treatment increased the derivation efficiency of pluripotent stem cells from cloned embryos using cryopreserved oocyte.

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.

Endogenous Retroviruses Function as Gene Expression Regulatory Elements During Mammalian Pre-implantation Embryo Development.

Pre-implantation embryo development encompasses several key developmental events, especially the activation of zygotic genome activation (ZGA)-related genes. Endogenous retroviruses (ERVs), which are regarded as “deleterious genomic parasites”, were previously considered to be “junk DNA”. However, it is now known that ERVs, with limited conservatism across species, mediate conserved developmental processes (e.g., ZGA). Transcriptional activation of ERVs occurs during the transition from maternal control to zygotic genome control, signifying ZGA. ERVs are versatile participants in rewiring gene expression networks during epigenetic reprogramming. Particularly, a subtle balance exists between ERV activation and ERV repression in host–virus interplay, which leads to stage-specific ERV expression during pre-implantation embryo development. A large portion of somatic cell nuclear transfer (SCNT) embryos display developmental arrest and ZGA failure during pre-implantation embryo development. Furthermore, because of the close relationship between ERV activation and ZGA, exploring the regulatory mechanism underlying ERV activation may also shed more light on the enigma of SCNT embryo development in model animals.

Effect of supplemetation of Zebularine and Scriptaid on efficiency of in vitro developmental competence of ovine somatic cell nuclear transferred embryos

Somatic cell nuclear transfer (SCNT) technology has been applied in the construction of disease model, production of transgenic animals, therapeutic cloning, and other fields. However, the cloning efficiency remains limited. In our study, to improve SCNT efficiency, brilliant cresyl blue (BCB) staining were chosen to select recipient oocytes. In addition, DNA methyltransferase inhibitor Zebularine (5 nmol/L) and histone deacetylase inhibitor Scriptaid (0.2 μmol/L) were jointly used to treat sheep donor cumulus cells and reconstructed embryo. Moreover, the expression levels of embryonic development-related genes (OCT4, SOX2, H19, IGF2 and Dnmt1) of reconstructed embryo were also detected. Using BCB + oocytes as recipient cell, donor cumulus cells and reconstructed embryos were treated with 5 nmol/L Zebularine and 0.2 μmol/L Scriptaid, the blastocyst rate in Zeb + SCR-SCNT group (28.25%) was significantly higher than SCNT (21.16%) (p < 0.05). Furthermore, results showed that expression levels of OCT4, SOX2, H19, IGF2 and Dnmt1 genes in Zeb + SCR-SCNT embryos were more similar to IVF embryos. Our study proved that 5 nmol/L Zebularine and 0.2 μmol/L Scriptaid treating with sheep donor cumulus cells and reconstructed embryos improved SCNT blastocyst rate and relieve the abnormal expression of embryonic developmental related genes.

Production of Transgenic Porcine Embryos Reconstructed with Induced Pluripotent Stem-Like Cells Derived from Porcine Endogenous Factors Using piggyBac System.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.

Inhibition of RNA synthesis during Scriptaid exposure enhances gene reprogramming in SCNT embryos.

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Improves the In Vitro Developmental Competence and Reprogramming Efficiency of Cloned Bovine Embryos by Additional Complimentary Cytoplasm.

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.

Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos.

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.