scholarly journals 238.Determination of differentially displayed oxygen-sensitive genes in bovine blastocysts

2004 ◽  
Vol 16 (9) ◽  
pp. 238
Author(s):  
A. J. Harvey ◽  
M. Kirstein ◽  
A. Navarrete-Santos ◽  
K. L. Kind ◽  
B. Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Developmental Competence ◽  
Myelogenous Leukemia ◽  
Bovine Embryo ◽  
Nadh Dehydrogenase Subunit 2 ◽  
Regulated Gene Expression

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) oxygen moderate changes in gene expression are observed in the bovine blastocyst, compared with 3- to 4-fold increases in the mouse. We have determined that these moderate gene expression changes are most likely regulated by Hypoxia-Inducible Factor (HIF)-2 transcription factor activity in the bovine, in the absence of HIF1, although HIF2 target genes are largely unknown. The aim of this study was to screen, by differential display RT-PCR, for putative oxygen-regulated transcripts that might confer developmental competence in blastocysts cultured under varying oxygen atmospheres post compaction. In vitro-produced bovine blastocysts were generated using standard protocols. Compact morulae were randomly allocated to treatments under either 2%, 7% or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent and samples were reverse transcribed using Superscript II. cDNA was amplified using 10-mer primers in reactions containing 32Pα-labelled dCTP. Resulting bands were detected by autoradiography, excised, purified and ligated into pGEMT vectors for transformation and sequencing. Seven clones were identified as having high homology with known sequences in GenBank. Real-time PCR was undertaken to confirm oxygen-regulation using Sybr green master mix. Myotrophin mRNA was significantly increased following 2% oxygen culture, compared with 20% cultured blastocysts (P�<�0.01), as was GLUT1 (P�<�0.01). The expression of anaphase-promoting complex showed a significant association with oxygen, being higher in 2% cultured blastocysts (P�<�0.05). Acetyl-coA-acetyltransferase I, chronic myelogenous leukemia tumor antigen (CML66), cyclin I, NADH dehydrogenase subunit 2 and ribonucleotide reductase M1, genes identified using differential display, were not altered by post compaction oxygen concentration. This study has identified potentially HIF2-specific regulated genes, and supports the hypothesis that reduced oxygen concentrations post-compaction may influence bovine embryo development through oxygen-regulated changes in gene expression.

scholarly journals 221 TYPICAL HIF1-REGULATED GENES ARE UNALTERED BY OXYGEN IN BOVINE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 261
Author(s):  
A. Harvey ◽  
K. Kind ◽  
J. Thompson
Keyword(s):  
Gene Expression ◽  
Oxygen Concentration ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Expression Of Genes ◽  
Regulated Gene Expression

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) post-compaction oxygen conditions moderate changes in gene expression are observed in the bovine blastocyst (Harvey et al. 2004 Biol. Reprod. 71, in press), compared with 3–4 fold increases in the mouse (Kind et al. 2004 Mol. Reprod. Dev., in press). Specifically, GLUT-1 (Harvey et al. 2004), myotrophin, and anaphase-promoting complex 1 (Harvey et al., unpublished) mRNAs are increased in bovine blastocysts following 2% oxygen culture, compared with those cultured under 20% oxygen. These oxygen-mediated differences in gene expression in the bovine are most likely regulated by hypoxia-inducible factor (HIF)2 transcription factor activity, as we have previously observed that HIF1α protein is not detectable in bovine embryos whereas HIF2α is readily detectable (Harvey et al. 2004). The aim of this study was to determine the effect of post-compaction oxygen concentration on the expression of typically HIF1-regulated and potential HIF2-regulated (suggested from a mouse knockout study; Scortegagna et al. 2003 Nat. Genet. 35, 371) genes in bovine blastocysts. In vitro-produced bovine embryos were generated using standard protocols. Compact morulae were randomly allocated to treatments under 2%, 7%, or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and samples were reverse-transcribed using Superscript II (Invitrogen, Melbourne, Australia). Amplification and analysis of cDNA was achieved by real-time PCR using specific primers and Sybr green PCR master mix (Applied BioSystems, Melbourne, Australia). Statistically significant differences in gene expression were analyzed by ANOVA, P < 0.05. Examination of expression of genes known to be regulated by HIF1 in somatic cells (reviewed by Semenza 2002 Biochem. Pharm. 64, 993) revealed no oxygen-mediated alteration in expression of aldose reductase, cyclooxygenase 2, or inducible nitric oxide synthase. However, the expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001). Expression of glutathione peroxidase, and CuZn- and Mn-superoxide dismutase (putative HIF2-regulated genes) was not influenced by oxygen concentration post-compaction. This study suggests that typical HIF1-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo. These results complement previous observations that HIF1α protein is not detectable in blastocyst-stage bovine embryos, and suggest that LDHA may be an HIF2 target gene in the bovine embryo. As embryo development is influenced by oxygen concentration, levels of LDHA at the blastocyst stage may be used as a marker of oxygen responsiveness.

scholarly journals EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice

Blood ◽  
2015 ◽  
Vol 125 (18) ◽  
pp. 2845-2854 ◽  
Author(s):  
Hai Po H. Liang ◽  
Edward J. Kerschen ◽  
Irene Hernandez ◽  
Sreemanti Basu ◽  
Mark Zogg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Blood Coagulation ◽  
Target Genes ◽  
Adaptor Protein ◽  
Messenger Rnas ◽  
Initiation Complex ◽  
Induced Expression ◽  
Regulated Gene Expression

Abstract Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow–derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

120. OXYGEN REGULATED GENE EXPRESSION IN MOUSE CUMULUS CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 39
Author(s):  
K. Tam ◽  
K. Banwell ◽  
D. Froiland ◽  
D. Russell ◽  
K. Kind ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Oxygen Concentration ◽  
In Vitro Maturation ◽  
Target Genes ◽  
Cumulus Cells ◽  
Differentially Expressed ◽  
Oxygen Levels ◽  
Regulated Gene Expression

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that mediate the expression of a range of genes in response to low oxygen. Previously we showed that subsequent developmental outcomes were influenced by oxygen levels during in vitro maturation. The aim of the current study was to examine the effects of varying oxygen concentration during in vitro maturation of mouse COCs on expression of HIF target genes in the cumulus cells. I mmature COCs were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 d) and cultured for 17-18 h under 2, 5 or 20% O2. Hyaluronidase-treated and recovered cumulus cells were collected and mRNA extracted for analysis. A microarray approach (Affymetrix 430_2) was used to identify genes in cumulus cells that were differentially expressed under varying oxygen concentrations (2, 5, 10 and 20%). This revealed 218 differentially expressed probes, of which 34 were up-regulated with decreasing oxygen levels. The great majority of these were classified as HIF-regulated genes. Specific analysis from real time RT-PCR of HIF regulated target genes Slc2a1, Ldha, Pgk1, Eno1, Ndrg1, Bnip3 were all significantly up-regulated (by at least 5–fold) when cells were cultured at 2% or 5% oxygen, when compared to 20% oxygen. Hif-1a mRNA decreased when cumulus cells were cultured in 2%, compared to 20% oxygen. This study demonstrates that cumulus cell gene expression is influenced by oxygen concentration, and suggests that these effects are mediated by the HIF transcription factors.

scholarly journals New Target Genes Controlled by the Bradyrhizobium japonicum Two-Component Regulatory System RegSR

2007 ◽  
Vol 189 (24) ◽  
pp. 8928-8943 ◽  
Author(s):  
Andrea Lindemann ◽  
Annina Moser ◽  
Gabriella Pessi ◽  
Felix Hauser ◽  
Markus Friberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Bradyrhizobium Japonicum ◽  
Target Genes ◽  
Regulatory Gene ◽  
Wild Type ◽  
Transcription Profiling ◽  
Two Component ◽  
Regulated Gene Expression

ABSTRACT RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and ΔregR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of ΔregR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

scholarly journals Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 367-377 ◽  
Author(s):  
Sandra Milena Bernal ◽  
Julia Heinzmann ◽  
Doris Herrmann ◽  
Bernd Timmermann ◽  
Ulrich Baulain ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Global Methylation ◽  
Oocyte Developmental Competence

SummaryCyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

scholarly journals Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 551-564 ◽  
Author(s):  
N Ghanem ◽  
D Salilew-Wondim ◽  
A Gad ◽  
D Tesfaye ◽  
C Phatsara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Placental Development ◽  
Similar Expression ◽  
Maternal Interaction

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30–40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60–70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

Bmi1 Is Essential for Leukemic Reprogramming of Myeloid Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1423-1423
Author(s):  
Jin Yuan ◽  
Masahiro Takeuchi ◽  
Hideyuki Oguro ◽  
Masamitsu Negishi ◽  
Hitoshi Ichikawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Target Genes ◽  
Expression Profiles ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Immortalized Cells

Abstract Abstract 1423 Poster Board I-446 The polycomb group (PcG) protein Bmi1 plays an essential role in the maintenance of self-renewing hematopoietic stem cells (HSCs). Derepressed p16Ink4a and p19Arf are tightly associated with a loss of self-renewing capacity of HSCs in Bmi1-deficient mice. Deletion of both Ink4a and Arf genes substantially restores the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 maintains HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent senescence pathway. Meanwhile, Bmi1 was originally identified as a collaborating oncogene in the induction of lymphoma and was subsequently reported to be overexpressed in various human cancers including leukemia. Recent studies have demonstrated that PcG proteins bind to multiple regions of the genome and regulate a bunch of target genes. Therefore, we asked whether Bmi1 is essential for leukemic stem cells (LSCs) and tried to identify critical target genes for Bmi1 other than Ink4a and Arf in leukemia. We expressed the MLL-AF9 leukemic fusion gene in purified Lin−Sca-1−c-Kit+CD34+FcγRII/ IIIhi granulocyte/macrophage progenitors (GMPs) from wild-type, Bmi1−/−, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− mice and performed in vitro myeloid progenitor replating assay. GMPs from 4 different genetic backgrounds were all immortalized in vitro, although Bmi1-deficient cells showed a slightly decreased replating efficiency. We then infused the immortalized cells into lethally irradiated recipient mice. Mice infused with wild-type and Ink4a-Arf−/− cells developed acute myelogenous leukemia (AML) at 30 to 60 days after infusion. Mice infused with Bmi1−/− cells did not develop leukemia at all. While a significant portion of mice infused with Bmi1−/−Ink4a-Arf−/− cells developed AML, although they took much longer time compared to those mice infused with wild-type and Ink4a-Arf−/− cells. These results indicate that as in HSCs, the Ink4a /Arf locus is one of the major targets for Bmi1 in leukemogenesis. In order to find unknown targets of Bmi1 in LSCs, we compared gene expression profiles of purified c-KithiFcRγII/IIIhiCD34+ cells from Ink4a-Arf−/− and Bmi1−/−Ink4a-Arf−/− immortalized cells. We found that the loss of Bmi1 did not affect the induction of MLL-AF9 target gene expression. By contrast, a number of genes were derepressed in the absence of Bmi1. Among these, Tbx15, a transcriptional co-repressor gene, appeared to be regulated by Bmi1 and a potential tumor suppressor gene in the development of leukemia. Of interest, the majority of derepressed target genes in transformed Bmi1−/−Ink4a-Arf−/− cells, including Tbx15, remained unchanged by re-expression of Bmi1. Correspondingly, re-introduction of Bmi1 to transformed Bmi1−/−Ink4a-Arf−/− cells failed to rescue their compromised leukemogenic activity in vivo. Our findings suggest that Bmi1 is required for faithful epigenetic reprogramming of myeloid progenitors into LSCs by leukemic fusions and contributes to establish LSC-specific transcriptional profiles to confer full leukemogenic activity on LSCs. Disclosures: No relevant conflicts of interest to declare.

Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock

Zygote ◽  
2016 ◽  
Vol 24 (5) ◽  
pp. 748-759 ◽  
Author(s):  
Krishna C. Pavani ◽  
Erica Baron ◽  
Pedro Correia ◽  
Joana Lourenço ◽  
Bruno Filipe Bettencourt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Embryo Development ◽  
Target Genes ◽  
Housekeeping Genes ◽  
Early Embryo ◽  
Developmental Competence ◽  
Nuclear Maturation

SummaryThree assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P < 0.05) with HS1 for maturated oocytes at 86.4 ± 4.3; 65.5 ± 0.7; 51.3 ± 0.9; 38.1 ± 1.9 and 36.3 ± 0.9, for control, 6 h, 12 h, 18 h and 24 h, respectively. For assays 2 and 3, results demonstrated that DNMT1, Cx43 and HSPA14 were down-regulated in the embryos produced in the warm with respect to the cold months (P < 0.05). A constant up- and down-regulation of DNMT1 and HSPA14 genes were observed in both HS-treated samples. Also, an inconsistent pattern of gene expression was observed in Cx43 and CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

scholarly journals 227 CONSTRUCTION OF STAGE-SPECIFIC cDNA MICROARRAY, AND ANALYSIS OF IN VITRO PRODUCED PRE-IMPLANATION STAGE BOVINE EMBRYOS FOR DEVELOPMENTAL COMPETENCE

2005 ◽  
Vol 17 (2) ◽  
pp. 264
Author(s):  
S. Mamo ◽  
C.A. Sargent ◽  
N.A. Affara ◽  
K. Wimmers ◽  
S. Ponsuksili ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Expression Profiles ◽  
Developmental Competence ◽  
Cdna Clones ◽  
Bovine Embryo ◽  
Single Experiment

Microarray technology currently has wide acceptance as a research tool in the study of gene expression profiling, mainly as a result of its use for monitoring the expression profiles of thousands of genes in a single experiment. However, its use in analyzing gene expression in the pre-implantation stage of bovine embryo development has been limited for reasons such as the large amount of RNA required and the lack of bovine specific cDNA clone collections (Smith L and Greenfield A 2003 Hum. Mol. Genet. 12, 1–8). In this study, with the objectives of producing pre-implantation-stage-specific bovine cDNA clones and examining the developmental competence, eighty-two selected target clones of pre-implantation-stage-specific genes were prepared and spotted on the glass slide. Embryos were produced in vitro and mRNAs were isolated from contrasting probes of good quality matured oocytes and blastocyst-stage embryos using a Dynabead mRNA isolation kit by following the manufacturer's instructions. First-strand cDNA syntheses were primed with T7 Oligo d(T)21 primer, followed by random primed second-strand syntheses using a DOP master kit (Roche Diagnostics, Mannheim, Germany) and global amplification using the same primers used for the first- and second-strand syntheses. In vitro transcription was performed to amplify the RNA by using the AmpliScribe T7 transcription kit (EPICENTRE Technologies, Oldendorf, Germany), and the amplified RNA (aRNA) was purified using a RNeasy Mini kit (Qiagen, Hilden, Germany). Finally, the results of different RNA amplifications (aRNA) were tested by hybridization on microarrays and also using real-time PCR techniques. With these analyses, the sufficiency of the yield and linearity of amplification procedures were confirmed. Three micrograms each of aRNA were labelled with Cy3 and Cy5 dyes and hybridized to the array. After overnight incubation at 42°C, the slides were sequentially washed and scanned using an ArrayWorx biochip reader (Applied Precision, Marlborough, UK), and quantifications as well as all analyses were carried out using different TIGR software modules (Saeed AI et al. 2003 Biotechniques 34(2), 374–378). Analyses of the results of repeated hybridizations showed that 35 genes (43%), which belong to different functional groups, were differentially expressed between the two stages. Further independent analyses using real-time PCR confirmed the results of 25 genes. Hence, it is possible to conclude that the established methods can be used for large scale gene expression analysis, and the identified genes can be potential candidates for characterizing developmental competence.

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