scholarly journals Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How

2020 ◽  
Vol 22 (1) ◽  
pp. 236
Author(s):  
Kilian Simmet ◽  
Eckhard Wolf ◽  
Valeri Zakhartchenko
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Global Dna Methylation ◽  
Evidence Based ◽  
Nuclear Reprogramming ◽  
Epigenetic Mechanisms ◽  
Animal Biotechnology ◽  
Donor Cells ◽  
Unique Model ◽  
Nuclear Transfer Embryo

The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner. In this review, we describe—based on the growing number of reports published during recent decades—in detail where, when, and how manipulations of the epigenome of donor cells and reconstructed SCNT embryos can be performed to optimize the process of molecular reprogramming and the outcome of nuclear transfer cloning.

scholarly journals Histone H3 lysine 27 trimethylation acts as an epigenetic barrier in porcine nuclear reprogramming

Reproduction ◽  
2016 ◽  
Vol 151 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Bingteng Xie ◽  
Heng Zhang ◽  
Renyue Wei ◽  
Qiannan Li ◽  
Xiaogang Weng ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone H3 ◽  
Ips Cells ◽  
Epigenetic Mark ◽  
Nuclear Reprogramming ◽  
Mammalian Development ◽  
H3k27me3 Level ◽  
Donor Cells ◽  
Induced Pluripotent

Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 μM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 μM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.

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.

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.

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

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
E. Lee ◽  
K. Song ◽  
Y. Jeong ◽  
S. Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Skin Fibroblasts ◽  
Miniature Pig ◽  
Donor Cells ◽  
Fetal Fibroblasts

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

322 POTENTIAL OF PRIMARY PORCINE KIDNEY CELLS FOR THE GENERATION OF TRANSGENIC PIGS VIA SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 308
Author(s):  
A. Wuensch ◽  
A. Richter ◽  
M. Kurome ◽  
B. Kessler ◽  
V. Zakhartchenko ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Homologous Recombination ◽  
Somatic Cell ◽  
Single Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Porcine Kidney ◽  
Cell Clones ◽  
Donor Cells ◽  
Additive Gene

The generation of genetically tailored pig models for biomedical research using somatic cell nuclear transfer (SCNT) is an efficient and precise approach, whereas the outcome is crucially dependent on the source of nuclear donor cells. Especially for site-directed mutagenesis by homologous recombination, including the generation of single cell clones, the demands on the target cells are high. Different primary cells used for SCNT have been tested for their efficiency in SCNT experiments, but further characterisation of the specific cell types, their morphology, proliferation, lifespan, and stability of karyotype is mostly lacking. We have evaluated the potential of 2 primary porcine kidney cell lines (PKC) isolated from juvenile pigs by a simple collagenase digestion and culture in collagen-coated dishes as cell source for SCNT, including their morphology, proliferation capacity, transfection efficiency, and capacity to support full-term development of SCNT embryos after additive gene transfer or homologous recombination. Single cell clones generated by subcloning of PKC at passage 3 showed different morphologies, proliferation rates, and lifespan, indicating that PKC culture is a mixed population of different types of fibroblasts and/or other cells types. The PKC could be maintained in culture for up to 71 passages without signs of senescence and decreased proliferation, exhibiting a stable karyotype containing 74% normal chromosome numbers (2N = 38) determined from metaphase spreads. In contrast, porcine fetal fibroblasts (PFF) and porcine ear fibroblasts (PEF) could be not be passaged more than 20 times. The calculation of growth curves at passage 4 to 5 showed that PKC exhibited a higher proliferation rate with a population doubling time of 16.6 to 18.4 h compared with PFF (23.2. h) and PEF (32.9 h). Furthermore the determination of the developmental competence after SCNT using PKC at passage 4 in 3 independent experiments and in vitro cultivation for 7 days resulted in a higher blastocyst rate (21%) compared with that in PFF (9.1%) and PEF (4.3%). The comparison of different transfection methods (lipofection, nanofection, conventional electroporation, nucleofection), using an expression vector for green fluorescent protein (GFP), showed that the NucleofectorTM technology gave the best results with transfection efficiencies of 70 to 89%, high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of stress. So far, around 150 cloned piglets using 18 different gene constructs have been produced using stable transfected PKC after additive gene transfer and targeting of 3 different loci. These findings demonstrate that among the 3 tested types of donor cells, PKC, PFF, PEF, primary PKC have outstanding potential for the production of genetically modified pigs by SCNT. This work is supported by the DFG (FOR535, FOR793), the Bayerische Forschungsstiftung, and Mukoviszidose e.V.

29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 126
Author(s):  
X. Yang ◽  
J. Mao ◽  
E. M. Walters ◽  
M. T. Zhao ◽  
K. Lee ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Natural Science ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Group ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

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