75 CHANGES IN GENE EXPRESSION PATTERN DURING PRE- AND PERI-IMPLANTATION STAGES IN BOVINE CLONED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 196
Author(s):  
L. l. Rodriguez-Alvarez ◽  
J. F. Cox ◽  
R. Einspanier ◽  
F. O. Castro
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
Rna Extraction ◽  
Gene Expression Pattern ◽  
Internal Standard ◽  
Fibroblast Cell ◽  
Trophoblastic Cells ◽  
Nanog Expression ◽  
Expression Of Genes

In ruminants after blastocyst formation and before implantation the embryo elongates drastically. This peri-implantation stage is characterized by active proliferation of trophoblastic cells and by changes in gene expression. In cloned embryos inappropriate expression of genes during early stages could be the cause of embryonic losses. The aim of this study was to compare the expression pattern of selected developmentally important genes in Day 7 embryos produced by somatic cell nuclear transfer with gene expression at Day 17 during elongation. IVF embryos at each stage were used for comparison. For analysis we used an RT-PCR approach (conventional + quantitative real-time PCR; qPCR). For nuclear transfer we used handmade cloning with an adult fibroblast cell line that previously yielded live cloned calves. Cells were confluent, non-serum starved in passage 4. Cloned embryos were cultured in SOF medium for 7 days in the well of the well (WOW) system. Semen from a proven bull was used for IVF and the presumptive zygotes were cultured under the same conditions as cloned embryos. At Day 7, embryos (cloned or IVF) were either transferred to recipients or pooled in tens for RNA extraction using NucleoSpin RNA XS Kit (Macherey-Nagel, Düren, Germany), converted to complementary (c)DNA and amplified with specific primers in PCR reactions. Transferred embryos were recovered at elongation stage (Day 17); their RNA was extracted and treated as above. The genes studied were NANOG, FGF4, OCT4, EOMES, IFNtau, and ACTB (internal standard for quantification). Data were analyzed by Kruskal Wallis test using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Development to blastocysts was 65% for clones and 32% for IVF embryos; 19 and 6 elongated embryos were flushed back, respectively. For gene expression analysis we used 5 pools of 10 blastocyts each and 12 (6 IVF; 6 cloned) elongated embryos with embryonic disc. At pre-implantation, we found OCT4, FGF4, IFNtau, and NANOG expressed and their relative levels were quantified (except for NANOG). Expression of OCT4 was higher in the cloned embryos; expression of EOMES was not detected at this stage. At Day 17, all 5 genes were expressed and quantified via qPCR, and interesting differences were found: relative levels of expression of FGF4 decreased in both types of embryos from Days 7 to 17, whereas those for IFNtau, NANOG, and EOMES increased significantly. Expression of OCT4 remained constant in IVF embryos but not in the cloned embryos (higher at Day 7). Our results provide an initial approach to the dynamic changes occurring in the expression of developmentally important genes in the transition from expansion to elongation. The decrease in FGF4 expression as well as the increase in EOMES and NANOG expression is a new finding that could shed light on the mechanisms of trophoblastic differentiation in bovine elongation.

Myelomatous Plasma Cells Display An Intermediate Gene Expression Pattern Between a Normal Plasma Cell and a Memory B Cell

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1892-1892
Author(s):  
Alicia Baez ◽  
Jose Ignacio Piruat ◽  
Teresa Caballero-Velazquez ◽  
María Victoria Barbado ◽  
Isabel Alvarez-Laderas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Survival ◽  
Expression Pattern ◽  
Plasma Cells ◽  
Gene Expression Pattern ◽  
Hierarchical Cluster ◽  
Differentially Expressed ◽  
Expression Of Genes ◽  
Healthy Donors

Abstract Introduction Memory B cells (MBCs) remain viable in a non-proliferative state for years. These cells express genes involved in cell survival and anti-apoptotic factors, while repress the expression of cell cycle regulatory genes. During their differentiation into plasma cells (PCs), these cells develop an opposite gene expression pattern, with a higher expression of genes implicated in cell proliferation and activation, and a lower expression of survival genes. In multiple myeloma (MM) the PCs accumulate into the bone marrow due, at least in part, to failure of pro-apoptotic mechanisms normally expressed in PCs. In the present study we analyzed the gene expression patterns of MBCs and PCs from healthy donors and patients with MM, in order to determine whether or not myelomatous PCs share characteristics of MBCs and/or normal PCs, and to identify possible genes related to the pathophysiology of the disease. Methods MBCs were obtained by immunomagnetic separation from buffy coats of 5 aged healthy donors. Likewise, PCs were isolated from bone marrow of 6 healthy donors and of 5 patients with MM. Using microarray techniques we analyzed the expression of 45000 genes in all samples. We performed unsupervised hierarchical cluster of gene expression data using the average linkage and the Euclidean distance. To identify differentially expressed genes among experimental groups we applied non-parametric Kruskal Wallis test. The differences in expression with a p value < 0.05 were considered significant. All analyses were performed using the Multi-experiment Viewer 4.7.1 software. Results From the hierarchical cluster obtained we clearly identified two groups, one which included normal PCs samples and the other one containing myelomatous PCs and MBCs. Interestingly, myelomatous PCs displayed intermediate features between MBCs and normal PCs (Figure 1). We found 5159 genes differentially expressed between normal and myelomatous PCs. Among these, we identified 3455 genes which displayed a similar expression pattern between MBCs and myelomatous PCs, including caspases inhibitors, MAP kinases, ubiquitins, transcription and translation factors. Conclusion Myelomatous PCs display an intermediate gene expression pattern between normal PCs and MBCs. These cells display high expression of genes involved in cell survival that should be normally inactivated in the transit of MBC to a normal PC, so that its expression pattern is closer to a MBC than a normal PC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 82 EFFECT OF HIGH FETAL CALF SERUM CONCENTRATION IN THE GENE EXPRESSION PATTERN OF IN VITRO PRODUCED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
E. S. Caixeta ◽  
R. R. Maziero ◽  
M. D. Guastali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Lipid Metabolism ◽  
Expression Pattern ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Gene Expression Pattern ◽  
Differentially Expressed ◽  
Bovine Embryos

Even though FCS provides energy substrates, amino acids, vitamins, growth factors, and heavy-metal chelators, its supplementation has been associated with several embryo abnormalities such as mitochondrial degeneration, metabolic deviations, excessive lipid accumulation, and decreased embryo survival after cryopreservation. The aim of the present study was to evaluate the effect of high FCS concentration in the gene expression pattern of in vitro-produced bovine embryos. Slaughterhouse ovaries were used to obtain oocytes (N = 360), which were matured and fertilized in vitro (Day 0). Presumptive zygotes were divided in 2 culture media: with low (SOFaa with 0.5% BSA and 2.5% FCS) or high (SOFaa with 0.5% BSA and 10% FCS) FCS concentration. Cleavage was evaluated on Day 3. Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2, and 90% N2). The produced blastocysts were placed in PBS solution and washed five times. A single blastocyst was frozen in a minimal volume of PBS and stored at –80°C until RNA extraction. Total RNA extraction was performed using the PicoPure RNA isolation Kit (Applied Biosystems®, Foster City, CA, USA). Extracted RNA was evaluated through 2100-Bioanalyzer (Agilent Technologies®, Palo Alto, CA, USA) and DNAse treated (Qiagen®, Valencia, CA, USA). RiboAmp RNA Amplification Kit (Applied Biosystems®) was used to amplify the RNA (T7 RNA polymerase-catalysed amplification reaction). The aRNA output was evaluated through NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA). A biotin-labelled cRNA and fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization (N = 3 per group) using GeneChip Bovine Genome Array (Affymetrix®). Following hybridization, probe arrays were washed, stained, and scanned. Microarray data analysis was performed in the software FlexArray 1.6.1.1. Genes with a fold change of at least 1.5 and a probability of P < 0.05 were considered differentially expressed. The data from in vitro embryo production were analysed through the PROC GLM (SAS Institute Inc., Cary, NC, USA). Cleavage rate (81.4 ± 1.5 and 85.5 ± 1.4) and blastocyst production (41.8 ± 2.4 and 47.2 ± 2.8) were not different (P > 0.05) between low and high FCS concentrations, respectively. A total of 40 genes were differentially expressed between low and high FCS concentration. A total of 28 genes were annotated, with 37 genes up-regulated and 3 genes down-regulated by high FCS concentration. The associated network functions of gene expression, RNA damage and repair, and post-transcriptional modification; and cell-to-cell signalling and interaction were generated by Ingenuity Pathway Analysis® (Redwood City, CA, USA). Differentially expressed genes involved in carbohydrate metabolism (GAPVD1, MGAT4A), lipid metabolism (ELOVL5), cellular assembly and organisation (EZR, LRP2), and cell death and survival (DRT8) were identified. In conclusion, high FCS supplementation was associated with different expression profiles of genes regulating carbohydrate and lipid metabolism, cellular assembly and organisation, and cell death and survival. The authors acknowledge support from FAPESP and LNBio-CNPEM.

scholarly journals 25 MATERNAL-TO-EMBRYONIC TRANSITION FOLLOWING NUCLEAR TRANSFER OR PARTHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 121
Author(s):  
T. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
I. Lagutina ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Nuclear Transfer ◽  
De Novo ◽  
Gene Expression Pattern ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Parthenogenetic Activation

The successful development of embryos generated by somatic cell nuclear transfer (SCNT) requires the ooplasm to reprogram the nucleus. This establishes the gene expression pattern necessary for full development by mechanisms that are currently being clarified. The ooplasm action on somatic nuclei shows many common aspects to the process that leads to the creation of a functional embryonic genome from the differentiated sperm and egg genomes. In order to investigate this aspect we studied a critical phase of early embryonic development: the maternal to embryonic transition (MET). We compared the pattern and level of gene expression between bovine embryos derived from in vitro fertilization (IVF), from nuclear transfer of adult fibroblasts (NT), or from parthenogenetic activation (PG). The study was performed in cattle because MET, in this species, occurs over four cell cycles, making it easier to detect even small deviations. Oocytes, matured for 22 h and fertilized in vitro or after cumulus removal, were enucleated and fused to fibroblast cells. Nuclear transfer and Met II oocytes were activated at 24-26 h of maturation with ionomycin (5 �M) for 5 min and 6DMAP (2 mM) for 4 h and then cultured in mSOFaa. Embryos were harvested at the required time for analysis at the 2-, 4-, 8-, and 16-cell; morula; and blastocyst stages and stored snap-frozen in a minimal volume of medium in groups of 5-10 embryos. Semiquantitative RT-PCR was used to study the expression of Nanog, Oct-4, Zar-1, and Par-3, because these genes are directly involved in early embryo development and have a specific expression pattern during MET. Data were analyzed with one-way ANOVA followed by Student-Newman-Keuls All Pairwise Multiple Comparison. No difference in pre-implantation development was observed among the three groups. The Nanog expression pattern was unchanged in all three groups, becoming detectable from the 8-16-cell stage onward. Oct-4 mRNA was detected at all stages in every group, but only in NT embryos did a significant increase occur at the 16-cell stage, suggesting the onset of an anticipated embryonic transcription. the Zar-1 expression pattern, with the characteristic de-novo transcription peak at the 4-cell stage, was observed in both IVF and NT embryos but not in PG embryos. In this group, Zar-1 mRNA levels were significantly higher at the 2- and 4-cell stage than in all of the following stages. The Par-3 gene showed the biggest differences among groups: IVF embryos expressed this gene from the 8-cell stage onward, whereas NT embryos showed high levels of Par-3 mRNA already at the 2-cell stage. Surprisingly, PG embryos showed no detectable Par-3 levels at any stages. The results indicate that, although in vitro development was not affected, gene-specific expression differences during MET occurred among groups. Relating the specific functions exerted by each of these genes in early development to the changes observed following the different manipulations provides useful data toward a better understanding of the role of these genes and of the mechanisms of nuclear reprogramming. This work was supported by FIRB RBNE01HPMX, FIRST 2004, and ESF-EuroStells.

scholarly journals A Scientific Note of Housekeeping Genes for the Primitively Eusocial bee Euglossa viridissima Friese (Apidae: Euglossini)

Sociobiology ◽  
2018 ◽  
Vol 65 (4) ◽  
pp. 766
Author(s):  
Samuel Boff ◽  
Anna Friedel ◽  
Anja Miertsch ◽  
J. Javier Quezada-Euàn ◽  
Robert J Paxton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reference Genes ◽  
Developmental Stages ◽  
Gene Expression Pattern ◽  
Housekeeping Genes ◽  
Expression Of Genes ◽  
Gene Expression Studies ◽  
The Stability ◽  
Primitively Eusocial

Studies on the expression of genes in different contexts are essential to our understanding of the functioning of organisms and their adaptations to the environment. Gene expression studies require steps of normalization, which are done using the stable expression pattern of reference genes. For many different eusocial bees reference genes have been discovered, but not for the primitively eusocial euglossine bees.We used available genomic resources of euglossine species and the gene information of Apis melliferato develop a set of reference genes for the primitive eusocial bee Euglossaviridissima. We tested nine genes in distinct developmental stages three different algorithms to infer the stability of gene expression. The Tata binding protein(Tbp) and 14-3-3epsilon were the most stable genes across all different stages. The strongest deviation in gene expression pattern occurred in pupae, which require a different set of genes for normalizing gene expression. 

scholarly journals Transversal gene expression panel to evaluate intestinal health in broiler chickens in different challenging conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
L. Criado-Mesas ◽  
N. Abdelli ◽  
A. Noce ◽  
M. Farré ◽  
J. F. Pérez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Barrier Function ◽  
Nutrient Transport ◽  
Gene Expression Pattern ◽  
Vaccine Dose ◽  
Special Focus ◽  
Intestinal Health ◽  
Barrier Failure ◽  
Expression Of Genes

AbstractThere is a high interest on gut health in poultry with special focus on consequences of the intestinal diseases, such as coccidiosis and C. perfringens-induced necrotic enteritis (NE). We developed a custom gene expression panel, which could provide a snapshot of gene expression variation under challenging conditions. Ileum gene expression studies were performed through high throughput reverse transcription quantitative real-time polymerase chain reaction. A deep review on the bibliography was done and genes related to intestinal health were selected for barrier function, immune response, oxidation, digestive hormones, nutrient transport, and metabolism. The panel was firstly tested by using a nutritional/Clostridium perfringens model of intestinal barrier failure (induced using commercial reused litter and wheat-based diets without exogenous supplementation of enzymes) and the consistency of results was evaluated by another experiment under a coccidiosis challenge (orally gavaged with a commercial coccidiosis vaccine, 90× vaccine dose). Growth traits and intestinal morphological analysis were performed to check the gut barrier failure occurrence. Results of ileum gene expression showed a higher expression in genes involved in barrier function and nutrient transport in chickens raised in healthy conditions, while genes involved in immune response presented higher expression in C.perfringens-challenged birds. On the other hand, the Eimeria challenge also altered the expression of genes related to barrier function and metabolism, and increased the expression of genes related to immune response and oxidative stress. The panel developed in the current study gives us an overview of genes and pathways involved in broiler response to pathogen challenge. It also allows us to deep into the study of differences in gene expression pattern and magnitude of responses under either a coccidial vaccine or a NE.

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