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.

18 EMBRYO AGGREGATION IN PIG IMPROVES CLONING EFFICIENCY AND EMBRYO QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 139
Author(s):  
C. Buemo ◽  
A. Gambini ◽  
L. Moro ◽  
R. F. Y. Martin ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Dna Fragmentation ◽  
Embryo Development ◽  
Embryo Quality ◽  
T Test ◽  
Control Group ◽  
Reconstructed Embryo ◽  
Student’S T ◽  
Student’S T Test

In this study, we analysed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst size and cell number, DNA fragmentation levels by TUNEL assay, and the relative expression of genes associated with pluripotency, apoptosis, trophoblast markers, and DNA methylation in the porcine. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% polyvinyl alcohol) by a DC pulse of 1.2 kVcm for 80 μs. Then, embryos were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of zona-free embryos was achieved in a well of wells system in 100 μL of SOF medium. Two experimental groups were used, one control group with a single reconstructed embryo per microwell (1×) and the other group placing 3 reconstructed embryo per microwell (3x aggregation group). Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality. Our results showed that aggregation of 3× embryos increased blastocyst formation rate and blastocyst size of pig cloned embryos (Fisher’s test P < 0.05 and Student’s t-test P < 0.05, respectively). The DNA fragmentation levels in 3× aggregated cloned blastocysts were significantly decreased compared to 1x blastocyst (Student’s t-test P < 0.05). Levels of Oct4, Klf4, Igf2, Bax, and Dnmt1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially nondetectable (Student’s t-test P < 0.05). Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Abstract 130: Hypercholesterolemia Suppresses Carotid Artery Endothelial NOS3 Expression via Epigenetic Mechanisms

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Alex Sotolongo ◽  
Yi-Zhou Jiang ◽  
John Karanian ◽  
William Pritchard ◽  
Peter Davies
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Endothelial Cells ◽  
Dna Methyltransferase ◽  
Control Group ◽  
High Cholesterol Diet ◽  
Cholesterol Diet ◽  
High Cholesterol ◽  
High Fat

Objective: One of the first clinically detectable changes in the vasculature during atherogenesis is the accumulation of cholesterol within the vessel wall. Hypercholesterolemia is characterized by dysfunctional endothelial-dependent vessel relaxation and impaired NOS3 function. Since DNA methylation at gene promoter regions strongly suppresses gene expression, we postulated that high-fat/high-cholesterol diet suppresses endothelial NOS3 through promoter DNA methylation. Methods: Domestic male pigs were fed control diet (CD) or isocaloric high fat and high cholesterol diet (HC; 12% fat and 1.5% cholesterol) for 2, 4, 8 or 12 weeks prior to tissue collection. Furthermore, to determine the effects of risk factor withdrawal, an additional group of swine received HC for 12 weeks and then CD for 8 weeks; a control group received HC continuously for 20 weeks. Endothelial cells were harvested from common carotid aorta. In parallel in vitro studies, cultured human aortic endothelial cells (HAEC) were treated with human LDL, GW3956 (LXR agonist) and RG108 (DNA methyltransferase [DNMT] inhibitor). In cells from both sources, DNA methylation at the NOS3 promoter was measured using methylation specific pyro sequencing, and endothelial gene expression was measured using RT PCR. Results: HC diet increased plasma cholesterol level from 75 mg/dl on CD to a plateau of about 540 mg/dl within 2 weeks. Endothelial NOS3 expression was significantly reduced (71±9 % of CD) after 4 weeks of HC, a level sustained at subsequent time points. Withdrawal of HC for 8 weeks did not recover NOS3 expression. After 12-week HC, the NOS3 promoter was hypermethylated. Withdrawal of HC did not reverse NOS3 promoter methylation. In vitro treatment of HAEC with human LDL (200 mg/dl total cholesterol) or GW3956 (5μM) suppressed NOS3 mRNA to 50% and 30% respectively, suggesting that LXR/RXR is involved in suppression of NOS3. Nitric oxide production was consistently suppressed by GW3959. Both could be reversed through inhibition of DNMTs by RG108. Conclusions: DNA methylation and LXR/RXR pathway can mediate the HC-suppression of endothelial NOS3. The study identifies novel pharmaceutical targets in treating endothelial dysfunction. Crosstalk between these pathways is under investigation.

scholarly journals Intravesical prostaglandin e2 effectiveness in the prevention of urinary retention after transvaginal reconstruction of the pubo-cervical fascia and short arm sling according to Lahodny: a prospective randomized clinical trial

2010 ◽  
Vol 14 (1) ◽  
pp. 15 ◽  
Author(s):  
G. QUADRI ◽  
N. NATALE ◽  
C. SPREAFICO ◽  
C. BELLONI ◽  
D. BARISANI ◽  
...  
Keyword(s):  
Prostaglandin E2 ◽  
Urinary Retention ◽  
Study Groups ◽  
T Test ◽  
Control Group ◽  
Fisher Exact Test ◽  
Test Results ◽  
Exact Test ◽  
Student’S T ◽  
Student’S T Test

Intravesical prostaglandin E2 is effective in the recovery of spontaneous voiding after transvaginal reconstruction of the pubocervical fascia and short arm sling according to Lahodny. The aim of the study was to compare the effects of intravesical prostaglandin E2 in the prevention of urinary retention after transvaginal reconstruction of the pubocervical fascia and short arm sling according to Lahodny. STUDY DESIGN: From November 1996 to June 1999 fifty women underwent the Lahodny procedure for moderate/severe cystocele and stress urinary incontinence. Women were randomly assigned to 1 of the 2 study groups: intravesical prostaglandin E2 versus controls. Data obtained were analyzed with the Student t test and the Fisher exact test. RESULTS: Two patients of the treatment group had to be excluded from the study, one because of the wrong measurement of the post-voidal residual volume and another due to a fastidious burning sensation which appeared immediately after prostaglandin instillation and required the suspension of the treatment. No other side effects such as nausea, vomiting, diarrhea or hyperthermia were observed. Patients who underwent the prostaglandin E2 treatment showed a recovery of spontaneous voiding after 7.9&plusmn;6.7 days, whereas this interval was significantly longer in the control group, being 12.9&plusmn;9.7 days (p=0.04, Two tailed Unpaired Student's T test). CONCLUSION: The effectiveness and the low associated morbidity mark the treatment with intravesical prostaglandin E2 useful in the recovery of normal voiding after transvaginal pubocervical fascia reconstruction and short arm sling with the procedure according to Lahodny.

scholarly journals Study of Relationship of Anemia among Edentulous Patients

2018 ◽  
Vol 5 (2) ◽  
pp. 105-108
Author(s):  
Lijo Isaac ◽  
A. P. Nirmal Raj ◽  
Reshma Karkera ◽  
R Naveen Reddy
Keyword(s):  
Nutritional Status ◽  
T Test ◽  
Dental Status ◽  
Adult Patients ◽  
Control Group ◽  
Age Group ◽  
Edentulous Patients ◽  
Student’S T ◽  
Relationship Of ◽  
Student’S T Test

Very little studies were done on relationship of the dental status and the nutritional status. The present study was done to study relation between edentulism and the presence of anemia. The study was included of 46 adult patients with edentulism and same numbers of patients were taken as controls. The results were tabulated and analyzed with the help of IBM SPSS statistics 20 using student’s t test. The hemoglobin levels were lower in the edentulous patients that that of the control group. The present study had shown that the nutritional status were poor resulting in anemia in case of edentulous patients as compared to control group with the same age group.  

scholarly journals Effects of early unilateral mandibular first molar extraction on condylar and ramal vertical asymmetry

2014 ◽  
Vol 08 (02) ◽  
pp. 178-183 ◽  
Author(s):  
Koray Halicioglu ◽  
Mevlut Celikoglu ◽  
Suleyman K. Buyuk ◽  
Ahmet E. Sekerci ◽  
Celal Candirli
Keyword(s):  
Asymmetry Index ◽  
T Test ◽  
Control Group ◽  
Missing Teeth ◽  
Anterior Crowding ◽  
Mandibular Asymmetry ◽  
Group I ◽  
Student’S T ◽  
Mandibular First Molar ◽  
Student’S T Test

ABSTRACT Objective: The objective of the following study is to investigate the mandibular vertical asymmetry in a group of patients with early unilateral mandibular first molar extractions. Materials and Methods: Mandibular asymmetry index measurements (condylar, ramal and condylar-plus-ramal) were performed on the panoramic radiographs of a study group including 51 patients (mean age: 18.60 ± 1.11 years) and a control group of 51 patients (mean age: 18.53 ± 1.29 years). Group I included patients with a unilateral mandibular first molar extracted before the age of 12 years. Group II included patients with no extractions and had excellent Class I relationships, no missing teeth and slight or moderate anterior crowding. A paired t-test was used to determine possible statistically significant differences between the sides for the measurements. Student's t-test was used for the comparison of asymmetry index values between the groups and genders. Results: No group showed statistically significant sex-or side-specific differences for posterior vertical height measurements. Condylar asymmetry index and ramal asymmetry index measurements were not statistically different between the groups, while condylar-plus-ramal asymmetry index (CRAI) measurements were statistically different between the groups (P = 0.019). Conclusions: A slight difference for CRAI value was found in patients with early unilateral mandibular first molar extractions.

scholarly journals Evaluating the Effect of Slow-Stroke Back Massage on the Anxiety of Candidates for Cataract Surgery

2019 ◽  
Vol 12 (2) ◽  
pp. 12-17
Author(s):  
Maryam Keramati, MD ◽  
Mohammad Sadegh Sargolzaei, MSc ◽  
Ali Moghadasi ◽  
Mohammad Hasan Basirinezhad, MSc ◽  
Reza Mohammadpourhodki
Keyword(s):  
Cataract Surgery ◽  
Intervention Group ◽  
T Test ◽  
Control Group ◽  
Paired Samples ◽  
Significant Difference ◽  
Student’S T ◽  
Dependent Samples ◽  
Student’S T Test ◽  
Back Massage

Background: The patients under cataract sur-gery often experience anxiety not only during the surgery, but also prior to the surgery.Purpose: We sought to determine the effects of slow-stroke back massage on anxiety in patients undergoing cataract surgery. Setting: The study was conducted in the Amiral-momenin Hospital of Zabol city, south-east of Iran.Participants: A total of 60 candidates of cataract surgery participated in the study.Research Design: The participants were ran-domly allocated to either control or intervention groups. The intervention group received slow-stroke back massages, while patients in control group received routine interventions.Intervention: The slow-stroke back massage was performed on the patients assigned to the interven-tion group. The intervention was performed in the morning of the surgery day at 30 minutes before the surgery. The researcher performed each mas-sage session in a sitting position. The duration of each massage session was 15 minutes. Main Outcome Measures: Anxiety was assessed in the both groups in the morning of the surgery, before and immediately after the intervention. In-dependent samples Student’s t test, paired samples Student’s t test, and chi-squared test were used to analyze the data.Results: Anxiety was not significantly different between the two groups before and after the mas-sage (p = .816). On the other hand, paired samples Student’s t test showed a significant difference comparing the anxiety scores before (49.7±5.43) and after (45.16±3.89) the massage in the interven-tion group (p < .001). Conclusions: Based on our results, slow-stroke back massage, which is a low-cost and safe method, reduced anxiety in patients who were candidates for cataract surgery.

scholarly journals In vitro study of marginal, internal and proximal adaptation of implant-supported single-crown CAD/CAM restorations

2020 ◽  
Vol 19 ◽  
pp. e207286
Author(s):  
Kamila Aguiar Figueiredo Alves ◽  
Janaina Emanuela Damasceno ◽  
Viviane Maia Barreto de Oliveira ◽  
Luiz Gustavo Cavalcanti Bastos ◽  
Andrea Nóbrega Cavalcanti
Keyword(s):  
Repeated Measures ◽  
T Test ◽  
Digital Impression ◽  
Single Crown ◽  
Significant Difference ◽  
Cad Cam ◽  
Student’S T ◽  
One Way Anova ◽  
Student’S T Test

Aim: This study evaluated the precision of a CAD/CAM system by measuring marginal, internal and proximal fits in implantsupported single-crown restorations. Methods: Ten models of the upper arch were made in which implants replaced the upper left premolars. For fabrication of the zirconia infrastructures, titanium bases (TiBase) were coded and scanned using a scan body. A second digital impression was made for the fabrication of prostheses. Silicone impression material was used to determine the internal clearance between the TiBase and infrastructure and between the infrastructure and crown, whose thickness was measured at three points [P1 (cervical), P2 (middle) and P3 (occlusal)] with a stereoscopic microscope at 70x and 100x magnification. One-way ANOVA for repeated measures and the Student t-test were used for the analysis of internal and marginal adaptation. Proximal contacts were analyzed qualitatively. Results: There was no significant difference between the teeth evaluated (Student’s t-test; p>0.05) or between the corresponding points evaluated in either tooth (one-way ANOVA; p>0.05). Analysis of the internal clearance between the infrastructure and crown demonstrated that all points were significantly different compared to the reference standardized at 100 μm (Student’s t-test p<0.0001). There was no significant difference between P1 and P2, with the thickness at these two points being lower than that obtained at P3 (one-way ANOVA, p<0.05). The proximal contacts did not coincide with the quality defined by the device. Conclusion: The system tested was unable to produce implantsupported single-crown ceramic restorations with marginal, internal and proximal fits matching the digital workflow, with the inferior fits requiring adjustment prior to cementation.

scholarly journals Artifact induction by endodontic materials

2019 ◽  
Author(s):  
Fernanda Cristina Sales Salineiro ◽  
Igor Publio Talamoni ◽  
Solange Kobayashi Velasco ◽  
Fabiana Mesquita Barros ◽  
Marcelo De Gusmão Paraíso Cavalcanti
Keyword(s):  
Region Of Interest ◽  
T Test ◽  
Control Group ◽  
Distribution Analysis ◽  
Subjective Analysis ◽  
Gutta Percha ◽  
Student’S T ◽  
The One ◽  
Student’S T Test

Metallic objects, such as intracanal posts and restorations, may produce severe interference, thus diminishing the quality of CBCT imaging. Objective: The purpose of this study was to analyze the influence of conventional and bioceramic gutta-percha points on the production of artifacts in CBCT images. Methods: Extracted single- -rooted premolar teeth (n=20) were instrumented and scanned with a CBCT device to create three groups: the Control group, the Gutta-Percha group and the Bioceramic Gutta-Percha group. Two types of analysis were executed: an objective one, using the Region of Interest (ROI) to measure the pixel density of each tooth, and a subjective one, to compare the groups’ images. For the statistical analysis, Student’s t-test, descriptive statistics and the frequency distribution analysis were used for both objective and subjective analyses. Results: The agreement between the observers ranged from moderate to excellent. Similar grayscale values were obtained in both the GP and BCGP groups. These results were endorsed by the p-values obtained with Student’s t test. For the subjective analysis, the observers indicated the BCGP group as the one that developed the highest number of artifacts. Conclusions: Both materials produced artifacts in the CBCT images. However, in the subjective analysis, the BCGP group showed higher levels of artifact production than the GP group, which could result in the misdiagnosis of root fracture and in a worse prognosis for that tooth.

129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 212
Author(s):  
G. Machado ◽  
A. Ferreira ◽  
I. Pivato ◽  
A. Fidelis ◽  
J. F. Srpicigo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
T Test ◽  
Molecular Profile ◽  
Control Group ◽  
Fluid Medium ◽  
Uterine Flushing

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality. Financial support: Embrapa, CNPq, CAPES.

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.

Sign in / Sign up

Export Citation Format

Share Document