scholarly journals Epigenetic abnormalities associated with somatic cell nuclear transfer

Reproduction ◽  
2021 ◽  
Author(s):  
Atsuo Ogura ◽  
Shogo Matoba ◽  
Kimiko Inoue
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mammalian Genome ◽  
Imprinted Genes ◽  
Donor Genome ◽  
Genome Wide ◽  
Donor Cells ◽  
Genomic Reprogramming

Twenty-five years have passed since the birth of Dolly the sheep, the first mammalian clone produced by adult somatic cell nuclear transfer (SCNT). During that time, the main thrust of SCNT-related research has been the elucidation of SCNT-associated epigenetic abnormalities and their correction, with the aim of improving the efficiency of cloned animal production. Through these studies, it has become clear that some epigenomic information can be reprogrammed by the oocyte, while some cannot. Now we know that the imprinting memories in the donor genome, whether canonical (DNA-methylation-dependent) or noncanonical (H3K27me3-dependent), are not reprogrammed by SCNT. Thus, SCNT-derived embryos have the normal canonical imprinting and the erased noncanonical imprinting, both being inherited from the donor cells. The latter can cause abnormal phenotypes in SCNT-derived placentas arising from biallelic expressions of noncanonically imprinted genes. By contrast, repressive epigenomic information, such as DNA methylation and histone modifications, might be more variably reprogrammed, leaving room for technical improvements. Low-input analytical technologies now enable us to analyze the genome of gametes and embryos in a high-throughput, genome-wide manner. These technologies are being applied rapidly to the SCNT field, providing evidence for incomplete reprogramming of the donor genome in cloned embryos or offspring. Insights from the study of epigenetic phenomena in SCNT are highly relevant for our understanding of the mechanisms of genomic reprogramming that can induce totipotency in the mammalian genome.

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

2019 ◽  
Vol 31 (1) ◽  
pp. 148
Author(s):  
Y. H. Zhai ◽  
X. L. An ◽  
Z. R. Zhang ◽  
S. Zhang ◽  
Z. Y. Li
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chromatin Modification ◽  
Blastocyst Stage ◽  
Imprinted Genes ◽  
Cell Stage ◽  
Genomic Methylation

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P<0.05). The imprinted genes were significantly up-regulated (P<0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P<0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P<0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P<0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

2009 ◽  
Vol 21 (1) ◽  
pp. 113 ◽  
Author(s):  
C. Couldrey ◽  
M. P. Green ◽  
D. N. Wells ◽  
R. S. F. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cpg Island ◽  
Somatic Cells ◽  
Cpg Sites ◽  
Donor Cells ◽  
Genomic Regions ◽  
Methylation Patterns

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P < 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

scholarly journals S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 815-828 ◽  
Author(s):  
Byeong-Gyun Jeon ◽  
Gianfranco Coppola ◽  
Steven D Perrault ◽  
Gyu-Jin Rho ◽  
Dean H Betts ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
X Chromosome ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Telomerase Activity ◽  
X Chromosome Inactivation ◽  
Dna Demethylation ◽  
Chromosome Inactivation ◽  
Donor Cells

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment withS-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (∼1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes,ANT3,MECP2,XIAP,XIST, andHPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.

22 OXAMFLATIN TREATMENT ENHANCES NUCLEAR REPROGRAMMING BY INHIBITING XIST EXPRESSION AND REDUCING DNA METHYLATION IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
J. Mao ◽  
M. T. Zhao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
K. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Cell ◽  
Real Time ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Xist Gene ◽  
Nuclear Reprogramming ◽  
Xist Expression ◽  
Donor Cells

Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) enhances developmental potential by alteration of epigenetic status. Oxamflatin is one of the potent HDACi. In our previous study, development to Day 7 blastocysts was enhanced when the porcine somatic cell nuclear transfer (SCNT) embryos were treated with oxamflatin for 16 h. The objective of the present study was to investigate the effect of oxamflatin treatment on XIST gene expression and DNA methylation of XIST gene and centromeric repeat element in Day 7 SCNT blastocysts. Somatic cell nuclear transfer was performed on enucleated metaphase II oocytes using a transgene female cell line. Cloned embryos were electrically fused and activated, treated with 150 nM oxamflatin for 16 h and cultured in PZM3 under 5% CO2, 5% oxygen, and 90% N2 for 7 days. Clones without Oxamflatin treatment were used as controls. For XIST methylation, IVF blastocysts at Day 7 were used as controls. Blastocysts at Day 7 were pooled from each treatment group and processed for methylation analysis by bisulfite sequencing and gene expression by quantitative real-time PCR. This experiment was replicated 4 times. The percent of CpG methylation in donor cells before SCNT was also determined. Data were analysed by using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). In donor cells, 45.3 ± 5.8% of CpGs in a centromeric repeat element (9 CpGs in GenBank Z75640) were methylated. In the SCNT embryos, oxamflatin treatment reduced methylation from 27.3 ± 3.1% in the control to 18.2 ± 3.2% (P < 0.05). The average methylation in XIST (11 CpGs in GenBank KC149530.1) in donor cells was 42.4 ± 6.4%. This CpG island had 2 sites that were not methylated in any of the samples. However, the remaining 9 CpGs were methylated in 8 of 15 samples; for example, showing a parental imprint of ~50%. This implied that the CpG island studied represented the real-time status of the XIST locus in the cell and provides a good marker for reprogramming studies. XIST methylation level in Day 7 blastocysts was not different between oxamflatin (11.8 ± 3.2%) and control (11.8 ± 3.2%). However, XIST methylation in SCNT embryos was higher than in the same age IVF blastocysts (11.7 ± 1.7 v. 0.6 ± 2.4%; P < 0.01). Oxamflatin treatment tended to decrease XIST expression in Day 7 blastocysts compared with controls (18.8 ± 0.8 v. 21.7 ± 0.8; P < 0.1) as measured by real-time PCR. Interestingly, XIST gene expression was positively correlated with its methylation (P < 0.05). In conclusion, these results indicate that during nuclear reprogramming there was a dramatic decrease in DNA methylation from donor cells to Day 7 SCNT embryos. The higher methylation of XIST in SCNT embryos compared with IVF embryos suggests that the reprogramming of donor cells was not completed, which may be a contributor to low cloning efficiency. Oxamflatin treatment of SCNT embryos may enhance nuclear reprogramming by inhibiting XIST expression and reducing DNA methylation, resulting in better embryo development.

Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 357 ◽  
Author(s):  
Hui Cao ◽  
Jun Li ◽  
Wenlong Su ◽  
Junjie Li ◽  
Zhigang Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Control Group ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Pluripotency Genes

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P&lt;0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5nM zebularine for 12h compared with the control group (25.4±1.6 vs 11.8±1.7%, P&lt;0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P&lt;0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes

2013 ◽  
Vol 25 (8) ◽  
pp. 1142 ◽  
Author(s):  
Insung Hwang ◽  
Yeon Woo Jeong ◽  
Joung Joo Kim ◽  
Hyo Jeong Lee ◽  
Mina Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Canis Lupus ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fusion Rate ◽  
Domestic Dogs ◽  
Domestic Dog ◽  
Canis Lupus Familiaris ◽  
Cloning Efficiency ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature’s diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P < 0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones’ inheritance of maternal domestic dog mitochondrial DNA.

scholarly journals Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs

2018 ◽  
Vol 50 (4) ◽  
pp. 1376-1397 ◽  
Author(s):  
Yanhui Zhai ◽  
Zhiren Zhang ◽  
Hao Yu ◽  
Li Su ◽  
Gang Yao ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Modifications ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Dna Demethylation ◽  
Epigenetic Reprogramming ◽  
Expression Levels ◽  
Fetal Fibroblasts

Background/Aims: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. Methods: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells’ affects SCNT embryos development and the crosstalk between epigenetic signals. Results: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). Conclusion: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer

2015 ◽  
Vol 40 (1) ◽  
pp. 7-15
Author(s):  
Bo Fu ◽  
Liang Ren ◽  
Di Liu ◽  
Jian-zhang Ma ◽  
Tie-zhu An ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Live Donor ◽  
Donor Cells
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