Identification of Animals Produced by Somatic Cell Nuclear Transfer Using DNA Methylation in the Retrotransposon-Like 1 Promoter

2014 ◽  
Vol 16 (6) ◽  
pp. 411-417
Author(s):  
Christine Couldrey ◽  
Paul Maclean ◽  
David N. Wells
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer

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.

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos

2015 ◽  
Vol 17 (5) ◽  
pp. 404-414 ◽  
Author(s):  
Hongliang Sun ◽  
Fenghua Lu ◽  
Peng Zhu ◽  
Xiaohua Liu ◽  
Mingming Tian ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Histone Acetylation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer

scholarly journals Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts

2007 ◽  
Vol 75 (2) ◽  
pp. 250-264 ◽  
Author(s):  
Aaron J. Bonk ◽  
Rongfeng Li ◽  
Liangxue Lai ◽  
Yanhong Hao ◽  
Zhonghua Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Aberrant Dna Methylation

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

2008 ◽  
Vol 53 (13) ◽  
pp. 1996-2001 ◽  
Author(s):  
Jie Chen ◽  
DongJie Li ◽  
YanQin Liu ◽  
Cui Zhang ◽  
YunPing Dai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Methylation Status

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.

DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

2011 ◽  
Vol 13 (4) ◽  
pp. 307-314 ◽  
Author(s):  
Fei Gao ◽  
Shengting Li ◽  
Lin Lin ◽  
Jian Li ◽  
Yonglun Luo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Peripheral Blood Cells

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 125
Author(s):  
R. S. Deshmukh ◽  
O. Oestrup ◽  
E. Oestrup ◽  
M. Vejlsted ◽  
H. Niemann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Epifluorescence Microscopy ◽  
Preimplantation Embryos ◽  
Parthenogenetic Activation

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

36 SERIAL SOMATIC CELL NUCLEAR TRANSFER INCREASES PREGNANCY LOSSES IN GOATS

2017 ◽  
Vol 29 (1) ◽  
pp. 125
Author(s):  
M. Yang ◽  
J. Hall ◽  
Q. Meng ◽  
Z. Fan ◽  
I. Polejaeva
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Pregnancy Loss ◽  
Methylation Pattern ◽  
Fibroblast Cells ◽  
First Polar Body ◽  
Significant Difference ◽  
Dna Methylation Pattern

Serial cloning by somatic cell nuclear transfer (SCNT) has been successful in several mammalian species. This method can be beneficial for transgenic line expansion or resetting the lifespan of transgenic cells. Previous studies in bovine and porcine have shown a decrease in efficiency over multiple iterations of serial cloning. However, the contradictory data has been reported in mice where no decrease in cloning efficiency was observed after 25 generations of recloning. To our knowledge, no data have been reported investigating the efficiency of serial cloning in goats. The aim of this study was to evaluate whether there is an effect of recloning on goat SCNT efficiency. αMHC-TGF-β1 fetal fibroblast cells (containing transforming growth factor-β under control of a cardiac-specific promoter) were produced by electroporation and used for the first round of SCNT. For serial cloning, we used neonatal fibroblast cells obtained from skin biopsies used as nuclear donors. These cells were collected from the transgenic cloned goats generated by the first round of SCNT. Cumulus-oocyte complexes recovered from abattoir-derived ovaries using slicing technique were matured in vitro for 20 to 24 h. The first polar body and metaphase plate were removed from a cumulus cell-free oocyte, and a donor fibroblast cell was subsequently transferred into the enucleated oocyte. Fused embryos were then activated for 5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide. Activated embryos were cultured in G1 medium with 5 mg mL−1 BSA for 12 h, followed by surgical transfer into the oviducts of recipients synchronized to show oestrus within 12 h of SCNT. In total, 592 and 395 embryos were transferred to 37 and 25 recipient goats, respectively, for the first and second round of SCNT. Pregnancy rate, rate of pregnancy loss, and term rate were analysed by Chi-squared with a 2-tailed P-value. No significant difference was observed in Day 40 pregnancy rates (32.4 v. 36%) and term rates (32.4 v. 20%) between the first round of cloning and the successive recloning. However, the rate of pregnancy losses was significantly greater in recloning group (P < 0.05), with 4 out of 9 pregnancies lost between Day 40 of gestation and term, whereas no pregnancy losses were observed after Day 40 of gestation in the first-round cloning group. The greater pregnancy loss in the recloning procedure might be caused by accumulation of epigenetic errors resulting from incomplete reprogramming. We are assessing the DNA methylation pattern of differentially methylated regions (DMR) of 2 paternally imprinted genes (H19 and IGF2R) in the cloned and recloned goats and expect to see a difference in their imprinted gene DNA methylation pattern, which could explain the greater rate of pregnancy loss in recloned goats.

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