400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

26 Drugs that Modify Epigenetics...What do they do to Porcine Clones?

2018 ◽  
Vol 30 (1) ◽  
pp. 152
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
L. N. Moro ◽  
N. Canel ◽  
D. F. Salamone
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Electric Pulse ◽  
Donor Cell ◽  
T Test ◽  
Control Group ◽  
Developmental Rates ◽  
Student’S T ◽  
Student’S T Test

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. In this study, we assessed whether the use the inhibitor of DNA (cytosine 5) methyltransferase 5-azacitidine (5Aza) combined with the MEK inhibitor in the MAPK pathway PD0325901 (PD) could improve SCNT efficiency in pigs. In vitro maturation of cumulus–oocyte complexes was performed in TCM for 44 h at 39°C under 5% CO2. Cumulus cells and zona pellucida was removed from matured oocytes, followed by enucleation of the metaphase plate previously stained with Hoëchst 33342. Each enucleated oocyte was attached to a donor cell by phytohemagglutinin treatment followed by an electric pulse of 80V for 30 μs. After fusion, reconstituted embryos were activated by an electric pulse followed by an incubation in 2 mM 6-DMAP for 3 h. Cloned embryos were cultured in vitro in a modified well-of-well system in SOF medium, where 3 cloned embryos were placed per microwell (3X). The experimental group 3X + drugs was exposed for the first 3 days to 1 μM PD and 1 μM 5Aza in SOF medium. After washing, embryos were cultured until Day 7 in regular SOF medium. The control group (3X) was cultured in regular SOF medium for 7 days. In vitro embryo developmental rates, gene expression, histone acetylation, and DNA methylation status were studied. The use of epigenetic modifying drugs significantly increased blastocyst rates (40.9% v. 29%; Fisher’s test, P < 0.05) and embryo size (41.46% v. 28.56%; Student’s t-test, P < 0.05) compared with the control group. Regarding gene expression, an increase of the relative expression of genes related to cell differentiation (Igf2 and Cdx2), antiapoptotic pathways (Bcl-xl) and DNA methylation modulation (Mapk1) was observed (P < 0.05). Pluripotency genes Oct4 and Nanog did not show differences between groups. The Bax proapoptotic gene significantly decreased its expression after drug treatment, as did the Klf4 gene (P < 0.05). Results were analysed by Student’s t-test. According to Histone H3K27ac, which is associated with enhancers or gene promoters, its marker was located mainly in the nuclear periphery respect to the control group with a uniform dispersion, indicating that the treatment could be activating certain genes by locating them near the periphery. Histone H3K4me1 was more uniformly localised throughout the nucleus in both groups. The intensity of the fluorescence was measured by quantitative confocal microscopy using a histogram produced by the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Regarding DNA methylation by bisulphite sequencing, the 2 genes studied (Oct4 and DNMT1) showed a higher demethylation status for the treated group. Our results indicate that the combination of 5Aza+PD during early pre-implantation development dramatically increase blastocyst rates and embryo quality. This novel combination could be used as a strategy to improve the efficiency of SCNT in pigs and potentially other animals.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 184
Author(s):  
S. Canovas ◽  
E. Ivanova ◽  
S. Garcia-Martinez ◽  
R. Romar ◽  
N. Fonseca-Balvis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Profiles ◽  
Differential Expression Analysis ◽  
Gene Expression Pattern ◽  
Reproductive Technologies ◽  
Global Methylation ◽  
Species Specific

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P < 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

scholarly journals Hypermethylation in Calca Promoter Inhibited ASC Osteogenic Differentiation in Rats with Type 2 Diabetic Mellitus

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Lei Wang ◽  
Feng Ding ◽  
Shaojie Shi ◽  
Xingxing Wang ◽  
Sijia Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Methylation Level ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Target Fragment

The abnormal environment of type 2 diabetes mellitus (T2DM) leads to a substantial decrease in osteogenic function of stem cells. However, the gene sequence does not vary before and after disease for the patient. This phenomenon may be related to changes in osteogenesis-related gene expression caused by DNA methylation. In this study, we established T2DM models to extract adipose-derived stem cells (ASCs) for different gene identifications through DNA methylation sequencing. Specific fragments of methylation changes in the target gene (Calca) were identified by IGV analysis. CGRP was applied to compare the effects on ASCs-T2DM morphology via phalloidin staining, proliferation through CCK-8 assay, and osteogenic differentiation with osteogenic staining, qPCR, and repair of calvarial defect. Furthermore, 5-azacytidine (5-az) was used to intervene ASCs-T2DM to verify the relationship between the methylation level of the target fragment and expression of Calca. We found that the DNA methylation level of target fragment of Calca in ASCs-T2DM was higher than that in ASCs-C. CGRP intervention showed that it did not change the morphology of ASCs-T2DM but could improve proliferation within a certain range. Meanwhile, it could significantly enhance the formation of ALP and calcium nodules in ASCs-T2DM, increase the expression of osteogenesis-related genes in vitro, and promote the healing of calvarial defects of T2DM rat in a concentration-dependent manner. 5-az intervention indicated that the reduction of the methylation level in Calca target fragment of ASCs-T2DM indeed escalated the gene expression, which may be related to DNMT1. Taken together, the environment of T2DM could upregulate the methylation level in the promoter region of Calca and then decrease the Calca expression. The coding product of Calca revealed a promoting role for osteogenic differentiation of ASCs-T2DM. This result provides an implication for us to understand the mechanism of the decreased osteogenic ability of ASCs-T2DM and improve its osteogenic capacity.

scholarly journals Hairless is a nuclear receptor corepressor essential for skin function

2009 ◽  
Vol 7 (1) ◽  
pp. nrs.07010 ◽  
Author(s):  
Catherine C. Thompson
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Nuclear Receptors ◽  
Chromatin Remodeling ◽  
Transcriptional Repression ◽  
Critical Role ◽  
Epithelial Stem Cells ◽  
Skin Function

The activity of nuclear receptors is modulated by numerous coregulatory factors. Corepressors can either mediate the ability of nuclear receptors to repress transcription, or can inhibit transactivation by nuclear receptors. As we learn more about the mechanisms of transcriptional repression, the importance of repression by nuclear receptors in development and disease has become clear. The protein encoded by the mammalian Hairless (Hr) gene was shown to be a corepressor by virtue of its functional similarity to the well-established corepressors N-CoR and SMRT. Mutation of the Hr gene results in congenital hair loss in both mice and men. Investigation of Hairless function both in vitro and in mouse models in vivo has revealed a critical role in maintaining skin and hair by regulating the differentiation of epithelial stem cells, as well as a putative role in regulating gene expression via chromatin remodeling.

33 PRE-AND POST-IMPLANTATION DEVELOPMENT OF EMBRYOS CLONED FROM PORCINE SKIN-DERIVED SPHERE STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 97
Author(s):  
Y. H. Hao ◽  
D. Wax ◽  
Z. S. Zhong ◽  
C. N. Murphy ◽  
L. Spate ◽  
...  
Keyword(s):  
Stem Cells ◽  
Genetic Modification ◽  
Selection Process ◽  
Donor Cell ◽  
Porcine Skin ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Donor Cells ◽  
Post Implantation

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5�C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 � 8.2% (169/208) and 62.1 � 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 � 5.8% (53/208) and 25.2 � 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 � 1.9 (NT, maximum was 58) and 16.8 � 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.

25 CRYOPRESERVATION AND IN VITRO CULTURE AFFECT HISTONE ACETYLATION LEVELS OF BOVINE FIBROBLASTS

2008 ◽  
Vol 20 (1) ◽  
pp. 93 ◽  
Author(s):  
L. Chacón ◽  
M. C. Gómez ◽  
J. A. Jenkins ◽  
G. Wirtu ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Cell Line ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Donor Cell ◽  
Epigenetic Changes ◽  
Blastocyst Development ◽  
Histone 3 ◽  
Donor Cells

Long-term culture of bovine fibroblasts increases histone acetylation levels (Enright et al. 2003 Biol. Reprod. 69, 1525–1530), and blastocyst development and pregnancy rates are enhanced by using donor cells having high levels of histone acetylation (Yang et al. 2007 Reproduction 133, 219–230; Yang et al. 2006 Biol. Reprod. 76, 36–42). When histone acetylation levels are increased in amino acid residues, the nucleosome is less tightly bound to DNA and, as a consequence, relaxation of the chromatin structure occurs (Zlatanova et al. 2000 FASEB J. 14, 1697–1704). Exposure of the donor cell with relaxed chromatin structure to oocyte cytoplasm factors after nuclear transfer (NT) may be effective for nuclear reprogramming (Tóth et al. 2004 J. Cell Sci. 117, 4277–4287). Bovine embryos can be reconstructed with donor cells thawed immediately before NT, but it is not known if epigenetic changes arise during cell cryopreservation, and if NT efficiency is affected. The acetylated form of histone H3-lysine 9-lysine 14 (acH3K9/14) is associated with active chromatin configuration (Rice and Allis 2001 Curr. Opin. Cell Biol. 13, 263–273) and active transcription (Fuks 2005 Curr. Opin. Genet. Dev. 15, 490–495) and, thus, can be used as a marker to determine epigenetic changes in somatic cells. The purpose of our study was to evaluate histone acetylation levels of cultured and cryopreserved bovine fibroblasts. Cells were derived from skin of three adult cows and cultured in GMEM for 15 days. Fibroblasts from each of the three cell lines were analyzed at passage 1 (P1), 2 (P2), and 10 (P10). At each of the three passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days in culture during which time acetylation levels were measured in fresh and frozen cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated with accutase, resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA), and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). Then, cells were incubated with a fluorescein isothiocyanate (FITC) conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Overall, histone acetylation levels in frozen cells (70%) were lower than levels in fresh cells (86%; P < 0.05), and all cell lines, whether fresh or frozen, showed lower acetylation levels at P1 (61%) than at P2 and P10 (88% and 85%, respectively; P < 0.001). No significant differences were observed between individual cell lines for fresh and frozen treatments. However, frozen cells from cell line 3 showed lower histone acetylation levels than fresh cells from cell line 3 and cell lines 1 and 2 at each passage. In summary, histone acetylation levels were lower in cryopreserved bovine fibroblasts and were higher in cell lines cultured for longer times. Table 1. Flow cytometrically detected acetylation levels of lysine 9 on histone 3 in cultured or cryopreserved bovine fibroblast cell lines passed for 1, 2, or 10 times

30 EFFECT OF THE CYTOPLAST SOURCE AND KARYOPLAST TYPE ON THE DEVELOPMENT OF HANDMADE CLONED EMBRYOS IN GOATS

2012 ◽  
Vol 24 (1) ◽  
pp. 127 ◽  
Author(s):  
C. Feltrin ◽  
N. Mohamad-Fauzi ◽  
S. Gaudencio Neto ◽  
L. T. Martins ◽  
J. L. Almeida ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Size ◽  
Polar Body ◽  
Primary Cultures ◽  
Cumulus Cell ◽  
Donor Cell ◽  
Transgenic Goats ◽  
Handmade Cloning

The aim of this study was to compare the effect of 2 donor cell types (bone marrow-derived mesenchymal stem cells, BM-MSC and skin fibroblast cells, SFC) and the source of oocytes (in vivo- and in vitro-matured goat oocytes) on the developmental capacity of handmade cloned goat embryos, following our procedures adapted from cattle (Ribeiro et al., 2009, Cloning Stem Cells 11, 377–386). In vivo- and in vitro-matured oocytes obtained postmortem from 36 superovulated and 90 nonstimulated goats were used as cytoplasts for cloning, after 26 h from the induction of ovulation or 20 h from the onset of IVM, respectively. Subsequent to cumulus cell removal and polar body selection, a total of 242 in vivo- and 580 in vitro-matured oocytes were subjected to zona removal, bisection in cytochalasin B and screening under ultraviolet light. Enucleated hemi-cytoplasts were exposed to phytohemoagglutinin and adhered to a single somatic cell (BM-MSC or SF) and electrofused by two 1.2 kV cm–1 DC pulses for 20 μs. Cell primary cultures were established from lysozyme transgenic goats. Prior to cloning, cells between the 3rd and 8th passage and at 50 to 60% (BM-MSC) or >95% (SFC) confluence were evaluated for size and viability using the CountessTM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). Fused structures were activated in ionomycin/6-DMAP and in vitro-cultured in the well of the well system in SOFaa + 5% FCS + 0.2% BSA, at 38.5°C, in 5% CO2, 5% O2 and 90% N2, for 6 days. After 8 replications, fusion, cleavage (Day 2) and embryo developmental (Day 6) rates were compared by the χ2 test. Data obtained on cell size and viability were analysed by ANOVA (P < 0.05). Cell viability was similar between SFC (86.7 ± 2.2%) and BM-MSC (89.0 ± 2.2%). However, mean cell size was significantly smaller in SFC (14.4 ± 0.4 μm) than in BM-MSC (20.1 ± 0.4 μm). Cell size appeared to be associated with fusion efficiency because fusion rates were also significantly lower with SFC than with BM-MSC (Table 1). However, cell type or oocyte source did not affect any other parameter for embryo production by cloning between groups. A total of 63 compact morulas and blastocysts from both cell and oocyte types were transferred, in groups of 4 to 5 embryos, to 15 synchronous recipients. Pregnancy diagnosis is performed by ultrasonography on Days 28 to 32. Thus far, one pregnancy derived from an embryo reconstructed with in vivo-matured oocytes and BM-MSC was obtained out of 9 recipients that received 37 embryos from all treatment groups. Six recipients with 26 embryos transferred are still pending diagnosis. In conclusion, the handmade cloning procedure using in vivo- and in vitro-matured oocytes and BM-MSC and SFC appears to be an effective alternative for the production of transgenic goats. Table 1.In vitro development of goat embryos produced by handmade cloning using human lysozyme (hLZ) transgenic cell lines Funded by the RECODISA Project, FINEP/MCT/Brazil.

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.

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

Zygote ◽  
2010 ◽  
Vol 19 (3) ◽  
pp. 255-264 ◽  
Author(s):  
Liliana Chacón ◽  
Martha C. Gómez ◽  
Jill A. Jenkins ◽  
Staley P. Leibo ◽  
Gemechu Wirtu ◽  
...  
Keyword(s):  
Donor Cell ◽  
Blastocyst Stage ◽  
Cell Passage ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Histone 3 ◽  
Donor Cells ◽  
Genetic Source ◽  
Vitro Development

SummaryIn this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.

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