• Reproductive and Developmental Biology
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• 제35권1호
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• BMB Reports
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• 제49권4호
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• pp.197-198
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• 2016

Although three different research groups have reported successful derivations of human somatic cell nuclear transfer-derived embryonic stem cell (SCNT-ESC) lines using fetal, neonatal and adult fibroblasts, the extremely poor development of cloned embryos has hindered its potential applications in regenerative medicine. Recently, however, our group discovered that the severe methylation of lysine 9 in Histone H3 in a human somatic cell genome was a major SCNT reprogramming barrier, and the overexpression of KDM4A, a H3K9me3 demethylase, significantly improved the blastocyst formation of SCNT embryos. In particular, by applying this new approach, we were able to produce multiple SCNT-ES cell lines using oocytes obtained from donors whose eggs previously failed to develop to the blastocyst stage. Moreover, the success rate was closer to 25%, which is comparable to that of IVF embryos, so that our new human SCNT method seems to be a practical approach to establishing a pluripotent stem cell bank for the general public as well as for individual patients.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.229-234
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• 2005

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ for 4 h (immediate activation after injection; IAI), or cultured in vitro for $2\~3$ h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation $2\~3$ h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of $36.6\%\;(15/41),\;39.5\%\;(17/43)\;and\;46.3\%$ (25/54), respectively. However, in the ABI group, only one embryo ($1.8\%$, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage $(4.9\%\cdot2/41)$. However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제27권2호
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• pp.266-277
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• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

• 한국수정란이식학회지
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• 제26권1호
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.