Treatment of Donor Cells and Reconstructed Embryos with a Combination of Trichostatin-A and 5-aza-2′-Deoxycytidine Improves the Developmental Competence and Quality of Buffalo Embryos Produced by Handmade Cloning and Alters Their Epigenetic Status and Gene Expression

2017 ◽  
Vol 19 (3) ◽  
pp. 208-215 ◽  
Author(s):  
Monika Saini ◽  
Naresh L. Selokar ◽  
Himanshu Agrawal ◽  
Suresh Kumar Singla ◽  
Manmohan Singh Chauhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Developmental Competence ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Handmade Cloning

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2’-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos

2016 ◽  
Vol 28 (6) ◽  
pp. 824 ◽  
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Trichostatin A ◽  
Developmental Competence ◽  
Simultaneous Treatment ◽  
Altered Expression ◽  
Expression Levels ◽  
Histone 3 ◽  
Donor Cells

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50 nM) and 5azadC (7.5 nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

65 EFFECT OF TRICHOSTATIN A AND 5-AZA-2'-DEOXYCYTIDINE TREATMENT OF DONOR CELLS, FUSED EMBRYOS, OR BOTH ON THE DEVELOPMENTAL COMPETENCE, QUALITY, AND EPIGENETIC STATUS OF CLONED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 162
Author(s):  
M. Saini ◽  
N. L. Selokar ◽  
H. Agrawal ◽  
S. K. Singla ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Species Conservation ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Epigenetic Modifiers ◽  
Donor Cells ◽  
Significant Difference ◽  
Transgenic Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology in buffalo for multiplication of elite animals, species conservation, and production of transgenic embryos for therapeutic applications. However, the cloning efficiency obtained in this species is very low, which might be due to improper reprogramming of donor cells after SCNT. Treatment of donor cells or fused embryos or both with epigenetic modifiers might be a suitable approach to improve the ability of donor cells to be reprogrammed. The present study was aimed at examining the effects of treatment of donor cells (24 h before SCNT) or fused embryos (10 h post-electrofusion) or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence, quality, and epigenetic status of buffalo embryos produced by hand-made cloning (HMC) as described earlier (Saini et al. 2014 Reprod. Fertil. Dev. doi: 10.1071/RD14176). The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher’s least significant difference test. The blastocyst rate was significantly higher (P < 0.05) and the apoptotic index was significantly lower (P < 0.05) in embryos produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls (Table 1). However, the cleavage rate and the total cell number were not significantly different among all the groups. The global level of H3K18ac, examined by immunofluorescence staining, was higher (P < 0.05) and that of H3K27me3 was lower (P < 0.01) in blastocysts produced from donor cells or fused embryos or both treated with TSA + 5-aza-dC than that of controls. These results show that treatment of donor cells, fused embryos, or both with TSA + 5-aza-dC improves the developmental competence and quality, and alters the epigenetic status of buffalo embryos produced by HMC. However, the effects of treatment with these epigenetic modifiers on the pregnancy rate require further studies. Table 1.Effect of treatment of donor cells, fused embryos, or both with 50 nM TSA + 7.5 nM 5-aza-dC on the developmental competence and level of apoptosis in cloned embryos

49 TRANSCRIPT LEVEL OF mRNA IN BOVINE CLONED OR RE-CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
G. Jang ◽  
H. Y. Jeon ◽  
K. H. Ko ◽  
H. J. Oh ◽  
H. J. Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Developmental Competence ◽  
Gene Transcript ◽  
Embryo Viability ◽  
Guanine Nucleotide Binding Protein ◽  
Glut 1 ◽  
Small Nuclear Ribonucleoprotein ◽  
Donor Cells ◽  
Fetal Fibroblasts

Gene expression in embryos played important roles during preimplantation development and had the potential to be used as an indicator for embryo viability. The purpose of this study was (1) to compare the developmental competence of cloned, or re-cloned embryos (Experiment 1); (2) to analyze the transcripts level of the related implantation, metabolic, and imprinting genes in IVF, cloned and re-cloned embryos (Experiment 2). The SCNT was performed according to the established system in our laboratory (Theriogenology 2006 65, 1800–1812). For producing cloned embryos, fetal fibroblasts as donor cells were used and a viable cloned calf was born. Recloned embryos derived from ear fibroblasts of the cloned calf, genetically same with donor fetal donor cells, were produced. The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) for up to 7 days. The developmental competence up to blastocysts was observed under a microscope. The implantation (Bax, E-cad, If-tau, Hsp 70, Igf2r, and DNMT1), metabolic [LDHA (Lactate Dehydrogenase A), G6PD (Glucose-6 Phosphate Dehydrogenase), PGK (Phosphogycerate Kinase), Na/K ATPase, and Glut-1], and imprinting [GNAS (guanine nucleotide binding protein, alpha stimulating), UBE3a (ubiquitin protein ligase E3A), Mest (mesoderm specific transcript), SNRPN (small nuclear ribonucleoprotein polypeptide N), and Ndn (necdin)] genes were selected. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitive RT-PCR. In Experiment 1, development competence of SCNT pre-implantation embryo was not different between cloned or re-cloned embryos (26% vs. 22%). In Experiment 2, the relative expression of Bax, Hsp70, If-tau, and Igf2r transcript was not different in IVF, cloned, and re-cloned embryos. Expression of E-cad and DNMT1 was higher in re-cloned embryos than any other group. Transcripts levels of LDHA, Na/K ATPase, and Glut-1 showed the similar relative abundance in IVF, cloned and re-cloned embryos. Expression of G6PD and PGF was increased in re-cloned and cloned embryos, respectively. Compared to IVF and cloned embryos, in re-cloned embryos, relative abundance of related imprinting genes (Ube3a, Mest, SNRPN, and Ndn) was increased. In conclusion, this study demonstrated that, whereas developmental competence of cloned or re-cloned embryos was not different, gene transcript levels were observed differently. It was suggested that alteration of gene expression in the re-cloned embryos derived from the cloned calf, genetically same with initial donor cells, might have affected the fetal development and births of re-cloned offspring.

28 HIGH HYDROSTATIC PRESSURE IMPROVED DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES FOR HANDMADE CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 94 ◽  
Author(s):  
Y. Du ◽  
L. Lin ◽  
C. Pribenszky ◽  
M. Molnár ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Nuclear Transfer ◽  
Technical Assistance ◽  
Developmental Competence ◽  
Control Groups ◽  
Donor Cells ◽  
And Control ◽  
Handmade Cloning

High hydrostatic pressure (HHP) has been introduced into the field of embryology recently, with the possible mechanism that a sublethal HHP could induce the synthesis of molecular chaperons to protect the embryos from further stresses. Improved cryotolerance has been achieved successfully in HHP-treated mouse (Pribenszky 2005 Anim. Reprod. Sci. 87, 143–150) and bovine (Pribenszky 2005 Reprod. Domest. Anim. 40, 338) embryos, and the semen of bull (Pribenszky 2007 Reprod. Fertil. Dev. 19, 181–182) and boar (Pribenszky 2005 Reprod. Fertil. Dev. 18, 162–163). The objective of the present study was to apply this new technique to in vitro-matured (IVM) porcine oocytes and further investigate its effect in the procedure of handmade cloning (HMC). After 40 h IVM, cumulus–oocyte complexes (COCs) were loaded in 0.5-mL straws by a 2-mL syringe, with HEPES-buffered TCM199 as the loading medium. COCs were then treated with 20 MPa (200 times greater than atmospheric pressure) for 60 min by a pressurizing device (Cryo-Innovation Inc., Budapest, Hungary), with an interval of 120 min between HHP treatment and subsequent HMC. Two different cell lines (from Day 40 fetuses of Yucatan and Danish Landrace breeds (LW1-2)) were used as donor cells for nuclear transfer. A total of 592 reconstructed embryos were produced from both HHP-treated and control groups and were in vitro cultured for 6 days to evaluate the developmental competence through to blastocyst formation. The effect of donor cells on blastocyst development was also investigated. SPSS 11.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis; values with P < 0.05 were regarded as significant. Blastocyst rates of the different groups are shown in Table 1. Our results indicated that COCs treated with HHP had a much higher blastocyst rate than those untreated (P < 0.01) and this improvement was not affected by using different donor cells for nuclear transfer. In conclusion, the sublethal HHP treatment could improve the in vitro developmental competence of porcine IVM oocytes when they are used for HMC. Further in vivo experiments are required to investigate the long-term effect of HHP on embryo development. Table 1. Day 6 blastocyst rates of HHP-treated and control groups with different donor cells for nuclear transfer The authors thank Ruth Kristensen and Janne Adamsen for their help and excellent technical assistance.

scholarly journals Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos

Reproduction ◽  
2007 ◽  
Vol 134 (6) ◽  
pp. 781-787 ◽  
Author(s):  
Gabbine Wee ◽  
Jung-Jae Shim ◽  
Deog-Bon Koo ◽  
Jung-Il Chae ◽  
Kyung-Kwang Lee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Trichostatin A ◽  
Dna Methyltransferases ◽  
Epigenetic Reprogramming ◽  
Preimplantation Development ◽  
Developmental Competence ◽  
Epigenetic Mark ◽  
Satellite Sequence ◽  
Donor Cells

Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the α-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the α-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
Z. Mei-Ling ◽  
Z. Yun-Hai ◽  
T. Yong ◽  
L. Ya ◽  
C. Hong-Guo ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Donor Cell ◽  
Developmental Competence ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Different Origins ◽  
Vitro Development

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins

303 ESTABLISHMENT OF PREGNANCIES WITH HANDMADE CLONING PORCINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS CONTAINING AN ALZHEIMER'S DISEASE GENE

2008 ◽  
Vol 20 (1) ◽  
pp. 231 ◽  
Author(s):  
P. M. Kragh ◽  
J. Li ◽  
Y. Du ◽  
L. Lin ◽  
M. Schmidt ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neurodegenerative Disorder ◽  
Polar Body ◽  
Developmental Competence ◽  
Amyloid Precursor Protein Gene ◽  
Reconstructed Embryos ◽  
Swedish Mutation ◽  
Handmade Cloning

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD). In the present study, we established pregnancies after transfer of SCNT blastocysts produced by the handmade cloning (HMC) technique. The blastocysts were transgenic for a human gene, amyloid precursor protein gene with the 'Swedish mutation' (APPsw), causing AD. For transgenesis, minipig fibroblasts were transfected by lipofection with a vector containing the APPsw gene under control of the platelet-derived growth factor β promoter (PDGF-APPsw) and a neomycin-resistance selection gene. Neomycin-resistant colonies were isolated, expanded, analyzed, and used for HMC. Cumulus–oocyte complexes were aspirated from ovaries of slaughtered sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent to the PB was removed with a microblade. The cytoplasts were used as recipients for embryo reconstruction. Reconstructed embryos were produced by a 2-step fusion procedure. At the first step, 1 cytoplast was fused with 1 fibroblast in the absence of Ca2+. After 1 h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously in the presence of Ca2+, incubated in cytochalasin B and cycloheximide for 4 h, and then cultured in PZM-3 medium. The development of reconstructed embryos to the blastocysts stage was determined after 5, 6, or 7 days of in vitro culture. To investigate the in vivo developmental capacity, blastocysts were transferred surgically to synchronized recipients. When using PDGF-APPsw-transgenic minipig fibroblasts, the rate of blastocyst formation (mean � SEM) was 39 � 3% (164/424). In comparison, non-transgenic fibroblasts resulted in a blastocyst development of 36 � 7% (36/102). In 4 recipients that received an average of 54 Day 5, 6, and 7 PDGF-APPsw-transgenic blastocysts, 2 ongoing pregnancies were confirmed by ultrasonography, 1 pregnancy was lost, and 1 returned to estrus. The results show a high in vivo developmental competence of blastocysts produced after SCNT of PDGF-APPsw-transgenic minipig fibroblasts.

Sign in / Sign up

Export Citation Format

Share Document