Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos

2019 ◽  
Vol 31 (12) ◽  
pp. 1885
Author(s):  
Thiago V. Damasceno Teixeira ◽  
Richard C. Fry ◽  
Angus McKinnon ◽  
Kerri L. Fry ◽  
Jennifer M. Kelly ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Somatic Cells ◽  
Early Embryo ◽  
Nuclear Reprogramming ◽  
Cloning Efficiency ◽  
Histone 3 ◽  
Starting Point ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Developmental Rates ◽  
Low Efficiency

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.

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

2017 ◽  
Vol 41 (3) ◽  
pp. 1255-1266 ◽  
Author(s):  
Jun-Xue Jin ◽  
Sanghoon Lee ◽  
Anukul Taweechaipaisankul ◽  
Geon A. Kim ◽  
Byeong Chun Lee
Keyword(s):  
Dna Methylation ◽  
Formation Rate ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Histone 3 ◽  
Expression Of Genes ◽  
Epigenetic Remodeling ◽  
Low Efficiency ◽  
Vitro Development

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

scholarly journals Inhibition of Histone Methyltransferase, Histone Deacetylase, and β-Catenin Synergistically Enhance the Cardiac Potential of Bone Marrow Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-15
Author(s):  
Jinpu Yang ◽  
Keerat Kaur ◽  
John G. Edwards ◽  
Carol A. Eisenberg ◽  
Leonard M. Eisenberg
Keyword(s):  
Bone Marrow ◽  
Histone Deacetylase ◽  
Heart Development ◽  
Bone Marrow Cells ◽  
Trichostatin A ◽  
Histone Methyltransferase ◽  
Early Embryo ◽  
Sarcomeric Protein ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Cardiac Gene

Previously, we reported that treatment with the G9a histone methyltransferase inhibitor BIX01294 causes bone marrow mesenchymal stem cells (MSCs) to exhibit a cardiocompetent phenotype, as indicated by the induction of the precardiac markers Mesp1 and brachyury. Here, we report that combining the histone deacetylase inhibitor trichostatin A (TSA) with BIX01294 synergistically enhances MSC cardiogenesis. Although TSA by itself had no effect on cardiac gene expression, coaddition of TSA to MSC cultures enhanced BIX01294-induced levels of Mesp1 and brachyury expression 5.6- and 7.2-fold. Moreover, MSCs exposed to the cardiogenic stimulus Wnt11 generated 2.6- to 5.6-fold higher levels of the cardiomyocyte markers GATA4, Nkx2.5, and myocardin when pretreated with TSA in addition to BIX01294. MSC cultures also showed a corresponding increase in the prevalence of sarcomeric protein-positive cells when treated with these small molecule inhibitors. These results correlated with data showing synergism between (1) TSA and BIX01294 in promoting acetylation of lysine 27 on histone H3 and (2) BIX01294 and Wnt11 in decreasing β-catenin accumulation in MSCs. The implications of these findings are discussed in light of observations in the early embryo on the importance of β-catenin signaling and histone modifications for cardiomyocyte differentiation and heart development.

scholarly journals Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming

2019 ◽  
Author(s):  
RV Sampaio ◽  
JR Sangalli ◽  
THC De Bem ◽  
DR Ambrizi ◽  
M del Collado ◽  
...  
Keyword(s):  
Donor Cell ◽  
Early Embryo ◽  
Nuclear Reprogramming ◽  
Toxic Chemical ◽  
Somatic Nucleus ◽  
Epigenetic Marks ◽  
Early Embryos ◽  
Donor Cells ◽  
Reprogramming Efficiency ◽  
Developmental Rates

AbstractOrchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone metyltransferases EHMT1 and EHMT2. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 causes diverse modifications to the chromatin during early development, suggesting an intense epigenetic crosstalk during nuclear reprogramming.

scholarly journals Influence of Selected Model Parameters on the Electromagnetic Levitation Melting Efficiency

2021 ◽  
Vol 11 (9) ◽  
pp. 3827
Author(s):  
Blazej Nycz ◽  
Lukasz Malinski ◽  
Roman Przylucki
Keyword(s):  
Electromagnetic Levitation ◽  
Optimization Methods ◽  
Calculation Model ◽  
Model Parameters ◽  
Starting Point ◽  
Low Efficiency ◽  
The Relationship ◽  
Reactive Metals ◽  
Electromagnetic Levitation Melting ◽  
Metal Melting

The article presents the results of multivariate calculations for the levitation metal melting system. The research had two main goals. The first goal of the multivariate calculations was to find the relationship between the basic electrical and geometric parameters of the selected calculation model and the maximum electromagnetic buoyancy force and the maximum power dissipated in the charge. The second goal was to find quasi-optimal conditions for levitation. The choice of the model with the highest melting efficiency is very important because electromagnetic levitation is essentially a low-efficiency process. Despite the low efficiency of this method, it is worth dealing with it because is one of the few methods that allow melting and obtaining alloys of refractory reactive metals. The research was limited to the analysis of the electromagnetic field modeled three-dimensionally. From among of 245 variants considered in the article, the most promising one was selected characterized by the highest efficiency. This variant will be a starting point for further work with the use of optimization methods.

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