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Pigs are valuable models for human disease research due to their physiological similarities to humans, and somatic cell nuclear transfer (SCNT) is commonly used to generate such models. However, SCNT efficiency is limited by incomplete epigenetic reprogramming and insufficient zygotic genome activation (ZGA). Lycopene, a potent antioxidant carotenoid, was investigated for its potential to improve porcine embryo development during in vitro culture (IVC). Parthenogenetically activated (PA) and SCNT embryos were cultured with various lycopene concentrations, with 0.2 µM showing the most significant benefits. Lycopene treatment significantly improved 4–5-cell cleavage, blastocyst formation, trophectoderm, and total cell numbers, while reducing apoptosis. It also decreased reactive oxygen species (ROS), upregulated the expression of antioxidant enzyme-related genes ( CAT , SOD1 , SOD2 , and HO-1 ), and increased mitochondrial membrane potential and autophagy in 4-cell embryos. Epigenetically, lycopene reduced H3K4me3, H3K9me3, and 5mC levels and downregulated methyltransferase-related genes ( ASH2L , SUV39H2 , DNMT1 , DNMT3A , and DNMT3B ), while upregulating ZGA-related genes ( ZSCAN4 , UBTFL1 , SUPT4H1 , MYC , and ELOA ). These findings suggest that lycopene treatment during IVC enhances embryonic development by reducing ROS-related mitochondrial dysfunction, inducing autophagy, and improving nuclear reprogramming, thereby improving ZGA in porcine SCNT and PA embryos.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives—such as trichostatin A—characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1–7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2–7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8–10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs. Summary sentence Chlamydocin analogues, a novel family of inhibitors specific for class I and IIb HDACs, significantly improved the ability of mouse SCNT-derived embryos to produce offspring.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG -edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (−/−) heterozygous, bi-allelic (−/−) homozygous, and mono-allelic (−/+) disruptions in BLG . Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (−/−) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG -edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr.

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.

In this study, the effect of trichostatin A (TSA)-mediated epigenomic modulation of nuclear donor cells on the developmental potential of caprine somatic cell cloned embryos was examined. The enucleated -matured oocytes were subzonally injected with adult ear skin-derived fibroblast cells exposed or not exposed to TSA (at a concentration of 50 nM). The experiment was designed on the basis of three different approaches to TSA-dependent modulation of donor cell-descended genome: before being used for somatic cell nuclear transfer/SCNT (Group I); immediately after activation of nuclear-transferred (NT) oocytes (Group II); or combined treatment both before being used for SCNT and after activation of NT oocytes (Group III). In the control Group IV, donor cell nuclei have not been treated with TSA at any stage of the experimental design. In TSA-treated Groups I and II and untreated Group IV, cleavage activities of cloned embryos were at the similar levels (80.6%, 79.8% and 77.1%, respectively). But, significant difference was observed between Groups III and IV (85.3 vs. 77.1%). Moreover, in the experimental Groups I and III, the percentages of cloned embryos that reached the blastocyst stages remarkably increased as compared to those noticed in the control Group IV (31.2% vs. 36.7% vs. 18.9%, respectively). In turn, among embryos assigned to Group II, blastocyst formation rate was only slightly higher than that in the control Group IV, but the differences were not statistically significant (25.8% vs. 18.9%). To sum up, TSA-based epigenomic modulation of somatic cell-inherited nuclear genome gave rise to increased competences of caprine cloned embryos to complete their development to blastocyst stages. In particular, sequential TSA-mediated modulation of both nuclear donor cells and activated NT oocytes led to improvement in the blastocyst yields of cloned goat embryos, which can result from enhanced donor cell nuclear reprogrammability.

The term animal cloning refers to an asexual mean of reproduction to produce genetically identical copies of any animal without the use of sperm. In India, the cloning of buffalo is well established and clones of the Murrah, the best dairy breed of buffalo, have been produced. The most acclaimed example is the restoration of progeny-tested breeding bull by isolating somatic cells from frozen doses of semen, which were stored for more than a decade in the semen bank. Buffalo bull cloning is considered the best available option to reproduce declared proven bulls and their semen would contribute to accomplishing the demand of ever-growing frozen semen, which is the prime requirement of conventional breeding. This article highlights the importance of buffalo bull cloning and its current status in India.

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

In this study, we first investigated the effects of 3-methyladenine (3-MA), an autophagy inhibitor, and the inducer – rapamycin (RAPA) on the incidence of programmed cell death (PCD) symptoms during development of porcine somatic cell nuclear transfer (SCNT)-derived embryos. The expression of autophagy inhibitor mTOR protein was decreased in porcine SCNT blastocysts treated with 3MA. The abundance of the autophagy marker LC3 increased in blastocysts following RAPA treatment. Exposure of porcine SCNT-derived embryos to 3-MA suppressed their developmental abilities to reach the blastocyst stage. No significant difference in the expression pattern of PCD-related proteins was found between non-transfected dermal cell and transfected dermal cell groups. Additionally, the pattern of PCD in SCNT-derived blastocysts generated using SC and TSC was not significantly different, and in terms of porcine SCNT-derived embryo development rates and total blastocyst cell numbers, there was no significant difference between non-transfected cells and transfected cells. In conclusion, regulation of autophagy affected the development of porcine SCNT embryos. Regardless of the type of nuclear donor cells (transfected or non-transfected dermal cells) used for SCNT, there was no difference in the developmental potential and quantitative profiles of autophagy/apoptosis biomarkers between porcine transgenic and non-transgenic cloned embryos. These results led us to conclude that PCD is important for controlling porcine SCNT-derived embryo development, and that transfected dermal cells can be utilized as a source of nuclear donors for the production of transgenic cloned progeny in pigs.