65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 138
Author(s):  
K. M. Whitworth ◽  
J. Zhao ◽  
L. D. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Histone Deacetylase ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Hdac Activity

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.

5 TRANSCRIPTIONAL PROFILING OF PIG IN IN VIVO, IN VITRO-FERTILIZED, AND NUCLEAR TRANSFER-DERIVED BLASTOCYSTS AND THE DONOR SOMATIC CELL LINE

2008 ◽  
Vol 20 (1) ◽  
pp. 83
Author(s):  
K. M. Whitworth ◽  
L. D. Spate ◽  
R. Li ◽  
A. Rieke ◽  
D. M. Wax ◽  
...  
Keyword(s):  
Cell Line ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Reference Sample ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Electrical Activation ◽  
Shock Proteins

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix� 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

scholarly journals 231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

2004 ◽  
Vol 16 (2) ◽  
pp. 236 ◽  
Author(s):  
Z. Beyhan ◽  
N.L. First
Keyword(s):  
Gene Expression ◽  
Statistical Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Live Birth ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Gene Expression Patterns ◽  
Donor Cells

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.

65 NUCLEAR REPROGRAMMING OF PORCINE CELLS AND THEIR USE AS DONOR CELL FOR NUCLEAR TRANSFER AFTER TREATMENT IN XENOPUS EGG EXTRACTS

2007 ◽  
Vol 19 (1) ◽  
pp. 150 ◽  
Author(s):  
K. Miyamoto ◽  
M. Ohnuki ◽  
N. Minami ◽  
M. Yamada ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Revealing an adequate cell state for nuclear reprogramming is essential to achieve efficient production of cloned embryos and animals. Previous reports suggest that nuclei from undifferentiated cells such as blastomeres or embryonic stem cells can support efficient development of cloned embryos to term. In recent years, differentiated somatic cells are frequently used for donor cells because of ease of preparation and application for genetic modification. The efficiency of the somatic cell nuclear transfer (SCNT) is still extremely low. We hypothesized that somatic cells that had been reprogrammed to dedifferentiated states before SCNT might support higher developmental ability of SCNT embryos. To test this hypothesis, porcine fibroblast cells were treated with Xenopus egg extracts, and the extract-treated cells (ETCs) were used as donor cell for SCNT to examine their ability to support early embryonic development. Xenopus egg extracts were prepared from activated S-phase eggs. Porcine fibroblast cells (106/mL) were permeabilized by 500 ng mL-1 of Streptolysin O and were incubated in the egg extracts with the energy-regenerating system for 2 hours at 23�C. After the extract treatment, permeabilized membranes were resealed in DMEM containing 2 mM CaCl2. The ETCs were fused with porcine enucleated oocytes and simultaneously activated. The reconstructed embryos were cultured in PZM-3 medium for 7 days. All statistical differences were analyzed by ANOVA. Reprogramming of ETCs was evaluated on changes of chromatin states and gene expression. Chromatin-binding proteins of ETCs were separated and analyzed on SDS-PAGE. Some proteins were incorporated onto and/or released from chromatins after the extract treatment. Especially, Xenopus egg-specific linker histone B4 was assembled on chromatins. Non-permeabilized control cells did not show these protein exchanges. Deacetylation of histone H3 lysine9 was detected in half number of ETCs in an ATP-dependent manner. In contrast, a high population of histone H3-acetylated cells was observed in buffer-treated cells as well as cells before the extract treatment. The pluripotent marker gene expression, such as OCT4 and SOX2, was also observed in ETCs after culture. The gene expression of these genes was not detected in non-treated cells. These results indicate that the extract treatment induces or triggers a part of dedifferentiation of somatic cells. These ETCs were used as donor cell for SCNT, and reconstructed cloned embryos were cultured. SCNT embryos showed no significant difference in cleavage rates and developmental rates to the blastocyst stage (25%) compared with non-treated control cells (26%). However, the total cell number of embryos at the blastocyst stage was significantly higher in SCNT embryos from ETCs compared with those of control cells (62 � 7 vs. 43 � 2, respectively; P &lt; 0.05). These results indicate that the extract treatment before nuclear transfer may stimulate cell proliferation of SCNT embryos but not improve early development. More studies, however, are needed to investigate their developmental ability to term.

scholarly journals The effect of extract of Dunaliella salina L. on expression of anti-apoptotic BCL2 in Hela cell line

2021 ◽  
Vol 43 (2) ◽  
pp. 186-192
Author(s):  
Mahdie- Sadat Lajavardi ◽  
Mahsa Kavousi
Keyword(s):  
Gene Expression ◽  
Treatment Group ◽  
Hela Cell ◽  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Group ◽  
P Value ◽  
Hela Cell Line ◽  
Relative Expression

Background: After breast cancer, cervix neoplasm is the most common disease among young women. Nowadays, natural substances are used in the treatment of diseases, because of the known side effects of the chemical drugs. Dunaliella is a green alga that lives in the saltwater lakes of Iran and is abundant in antioxidant substances. This paper aimed to study the effect of Dunaliella extract on the expression of the anti-apoptotic BCL-2 gene in Hela cell line. Expression of this gene is increased during cancer. It is expected that gene expression will be reduced if the alga extract is effective. Methods: Hela was prepared from the Center for Genetic and Biological Reserves of Iran and cultured. After culture, cells were divided into two treatment and control groups. Different concentrations of algae extract were applied to the treatment group for 48 hours. Then its toxicity was measured using MTT assay and IC50 was determined. RNA was extracted from cells of two groups, to determine the relative amount of gene expression at a concentration of IC50. Then Real-time PCR was used. Results: The result of Real-time PCR showed that the relative expression of BCL-2, in treatment group cells that were affected by algae extract, was four times lower than the control group. Since the P-value is less than 0.05 (P-value = 0), this decrease is significant. Conclusion: After 48 hours, at a concentration of IC50 of algae extract, the relative expression of BCL-2 was four times lower than control group.

scholarly journals Extracellular matrix (ECM) and cell adhesion molecules (CAMs) gene expression in brain GD-10, hippocampal cell (mHT-22 Cell Line), human glioblastoma-astrocytoma (hLN-405), and rat glioma cell (rF98) using real time PCR.

2014 ◽  
Vol 10 (2) ◽  
Author(s):  
Yulia Irnidayanti ◽  
Win Darmanto
Keyword(s):  
Gene Expression ◽  
Cell Adhesion ◽  
Cell Line ◽  
Real Time ◽  
Real Time Pcr ◽  
Glioma Cell ◽  
Gene Copy ◽  
Human Glioblastoma ◽  
Rat Glioma ◽  
Hippocampal Cell

The aim of this study is to compare ECM and CAMs gene expression in hippocampal cell, glioblastoma-astrocytoma and glioma cell using real time PCR at gestation day of 10 (GD-10) Result of real time PCR showed that expression level of extracelluler matrix of vimentin was higher than the expression of fibronectin, Neural cell Adhesion molecule (Ncam), neurofilament high (Nfh), neurofilament medium (NFm), neurofilament low (Nfl) and tenascin. The expression of vimentin in the brain also shows the highest when compare to the expression in the human glioblastoma-astrocytoma (hLN-405), rat glioma cell (rF98) and mouse hippocampal cell line (mHT-22). The number of vimentin gene copy was 538.554, whereas in the culture of hLN-405 was 2.246.309, in culture of rF98 was 974.368 and mHT-22 was 1.542.529. These findings suggested that vimentin most dominant expressed compare to the another gen. Expression of vimentin was gradually lower from astrocyte cell (hLN-405 or human glioblastoma-astrocytoma), then in hippocampus cell,  glioma cell and embryonic brain cell. ________________________________________GRAPHICAL ABSTRACT

scholarly journals Promotion of extrinsic apoptosis pathway in HCT-116 human colorectal cancer cell line by sodium butyrate as histone deacetylase inhibitor

2020 ◽  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Cell Line ◽  
Real Time ◽  
Histone Deacetylase ◽  
Sodium Butyrate ◽  
Histone Deacetylase Inhibitor ◽  
Control Group ◽  
Deacetylase Inhibitor ◽  
Hct 116

Background: Colorectal cancer (CRC) has already been considered the fourth leading cause of mortality worldwide as the genes involved in apoptotic pathways and alterations of reversible epigenetic have an important role in the progression of CRC. Objectives: The current study aimed to evaluate the effect of sodium butyrate as a histone deacetylase inhibitor on the alterations of the gene expression of FAS, Fas ligand (FASL), Death receptor 4 in HCT-116 CRC cell line. Methods: HCT-116 cell line was cultured in Dulbeccoʼs Modified Eagle Medium. The cytotoxicity effect of sodium butyrate on HCT-116 was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide assay for three incubation times (i.e., 24, 48, and 72 h). The half-maximal inhibitory concentration (IC50) values were determined. The optimum concentration was within the range of 6.25-200 mM. The cellular ribonucleic acid was extracted, and complementary deoxyribonucleic acid was synthesized. Finally, the alterations of the gene expression of FAS, FASL, DR4, DR5, and TRAIL were assessed by real-time polymerase chain reaction (PCR). Results: The IC50 levels for three incubation times were 50, 12.5, and 6.25 mM, respectively. The obtained results of real-time PCR demonstrated a significant increase in the gene expression of TRAIL, DR5, and FAS in comparison to that of the untreated cells as the control group at the three incubation times. The DR4 gene expression significantly increased in comparison to that reported for the control group at 48 and 72 h of incubation. In addition, FASL gene expression remarkably decreased at the three incubation times. Conclusions: Sodium butyrate could show cytotoxicity effect on CRC cell lines through the induction of death receptors in the extrinsic apoptotic pathway. The obtained results of this study revealed that the optimum effect of sodium butyrate is an incubation time-dependent and concentration-dependent manner.

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