Intracellular gene expression. Analysis of RNA from mycobacteria in macrophages using RT-PCR.
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Animals↗
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Gene expression analysis using quantitative reverse transcription-polymerase chain reaction and a multispecific internal control.
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Animals↗
Quantitative aspect of gene expression analysis in mammalian oocytes and embryos.
In vitro produced embryos, due to their lower developmental potential when compared to the in vivo derived counterparts, have been currently subjected to an intensive scientific investigation. Qualitative as well as quantitative analyses of gene expression (reverse transcription-polymerase chain reaction; RT-PCR) belong to the powerful tools providing a wide spectrum of data on the quality of oocytes and embryos. The main research areas in this topic concern the following categories: oocyte quality, developmental competence of in vitro produced (IVP) embryos also with regard to their sex, embryo metabolism, gene expression levels, embryo manipulation and cloning. There are many methods that have been applied to study gene expression, but only some of them meet the requirement of analysing small cell samples which usually is the case with oocytes or early embryos. This paper deals with the description of the current methods commonly used for quantitative gene expression in mammalian oocytes and preimplantation embryos with a special attention paid to the real time PCR.
Animals↗
Effect of ionizing irradiation on human esophageal cancer cell lines by cDNA microarray gene expression analysis.
To provide new insights into the molecular mechanisms underlying the effect of irradiation on esophageal squamous cell carcinomas (ESCCs), we used a cDNA microarray screening of more than 4,000 genes with known functions to identify genes involved in the early response to ionizing irradiation. Two human ESCC cell lines, one each of well (TE-1) and poorly (TE-2) differentiated phenotypes were screened. Subconfluent cells of each phenotype were treated with single doses of 2.0 Gy or 8.0 Gy irradiations. After a 15 min incubation time-point, the cells were collected and analyzed. Compared with non-irradiated cells, many genes revealed at least 2-fold upregulation or downregulation at both doses in well or poorly differentiated ESCC cells. The common upregulated genes in well and poorly differentiated cell types at both irradiation doses included SCYA5, CYP51, SMARCD2, COX6C, MAPK8, FOS, UBE2M, RPL6, PDGFRL, TRAF2, TNFAIP6, ITGB4, GSTM3, and SP3 and common downregulated genes involved NFIL3, SMARCA2, CAPZA1, MetAP2, CITED2, DAP3, MGAT2, ATRX, CIAO1, and STAT6. Several of these genes were novel and not previously known to be associated with irradiation. Functional annotations of the modulated genes suggested that at the molecular level, irradiation appears to induce a regularizing balance in ESCC cell function. The genes modulated in the early response to irradiation may be useful in our understanding of the molecular basis of radiotherapy and in developing strategies to augment its effect or establish novel less hazardous alternative adjuvant therapies.
Carcinoma, Squamous Cell↗
Gene-expression analysis after alcohol exposure in the developing mouse.
Exposure to alcohol in the embryonic mouse can lead to structural and neurophysiologic changes. The cause of these changes is poorly understood, but they are likely the result of numerous mechanisms. Here we investigate ethanol-induced alterations in gene expression in the fetal brain. Using complementary-DNA microarrays, we identified 25 genes that were down-regulated by prenatal ethanol exposure on days 7 and 9 of gestation. None were found to be up-regulated. Of those that were repressed, 6 (Timp4, Bmp15, Rnf25, Akt1, Tulp4, Dexras1) have been identified, and they are discussed here in the context of the developing fetus. The identified genes have been shown to be involved in cell proliferation, differentiation, and apoptosis, and they contribute to tissue growth and remodeling, as well as neuronal growth and survival. Microarray studies may be useful in the identification of a genetic marker for fetal alcohol syndrome, the discovery of novel pathways that may be involved in its origin, or both.
Animals↗
Sequence analysis, gene expression, and chromosomal assignment of mouse Borg4 gene and its human orthologue.
The Borg (binder of Rho GTPases) family proteins interact with CDC42 and TC10 in a guanosine triphosphate (GTP)-dependent manner. We have isolated a full-length cDNA of the mouse Borg4 gene, which is a member of this family. Sequence analysis revealed that this gene encoded a putative 349-amino acid protein. By reverse transcription--coupled polymerase chain reaction (RT-PCR) analysis, we observed that Borg4 was expressed ubiquitously in adult tissues. Additionally, we determined the entire cDNA sequence of the putative human Borg4 orthologue. By fluorescence in situ hybridization, mouse Borg4 and the putative human orthologue have been assigned to mouse chromosome 11E and human chromosome 17q24-25, which has been described as syntenic to the mouse region.
Adaptor Proteins, Signal Transducing↗
Large scale gene expression analysis of CBA/J mouse strain fetal thymus using cDNA-array hybridizations.
The CBA/J inbred mouse strain constitutes an interesting in vivo model-system for studies on molecular genetics of thymus ontogeny. Using RT-PCR method we have found previously that several immune system related genes as interleukins and MHC are differentially expressed. During this period the onset of T-cell receptor beta rearrangements also occur. To know which other genes are modulated during the ontogeny of the thymus, the mRNA expression levels of fetal thymus (15 and 16 days gestation) of CBA/J mouse strain were measured by hybridization with a set of four macroarrays containing a panel of 6,144 IMAGE cDNA clones from MTB thymus library. We found 145 differentially expressed sequences; 44 were up- and 101 down-regulated in the thymus at 15-16 days gestation. Among these sequences, only 20 are identified as genes whose functions are known and 125 are still unknown. Our data demonstrated that, despite intense research on maturation of the immune system focusing on the activity of several well-characterized genes, the large scale expression profile during thymus ontogeny is still an open matter. The use of cDNA-array technology is an affordable method to identify new genes that may play a role in this phenomenon.
Animals↗
Gene expression analysis in the stomachs of water immersion-restraint stress rats using high-density oligonucleotide array.
BACKGROUND AND AIM: Research on gastric lesions developing in response to stress is essential to elucidating the pathogenesis of these lesions as well as the interplay with other factors, including Helicobacter pylori infection and non-steroidal anti-inflammatory drug use. Genes expressed individually or in sets, such as heat shock proteins, growth factors, proto-oncogenes and cyclooxygenases, have been investigated in the stomach. However, gene expression in the stomach after stress exposure have not yet been comprehensively examined. We investigated the gastric gene expression profile in response to stress. METHODS: A high-density oligonucleotide array, representing approximately 850 genes, was used to determine gene expression changes in the stomachs of water immersion-restraint stress (WIRS) rats. RESULTS: Fifty-eight genes including expressed sequence tag (EST) genes were upregulated more than twofold in the 30 min-WIRS rat stomach as compared with the control. Concomitantly, five genes were downregulated. Numbers of up- or downregulated genes decreased rapidly at 1 and 2 h of WIRS. Altered gene expression of heat shock proteins, cell cycle regulators, proto-oncogenes and metabolic enzymes were recognized. Several of these genes, including p38 mitogen-activated protein kinase, did not reportedly show gastric expression changes in response to stress. CONCLUSION: These results suggest that, in addition to the previously identified stress-induced genes, expression of a number of other genes in the stomach is also involved in stress response.
Animals↗
Gene expression analysis by signature pyrosequencing.
We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.
Base Sequence↗
Type IV phosphodiesterase inhibition in experimental allergic encephalomyelitis of Lewis rats: sequential gene expression analysis of cytokines, adhesion molecules and the inducible nitric oxide synthase.
Type IV phosphodiesterase inhibitors are able to suppress EAE. To investigate the effects of this therapy in the central nervous system, we serially analyzed from days 7 to 17 postinoculation the gene expression pattern of tumor necrosis factor (TNF), lymphotoxin, interferon-gamma, interleukin-1beta, the inducible nitric oxide synthase (iNOs), interleukin-10, the vascular cell adhesion molecule-1 (VCAM-1) and the intercellular adhesion molecule-1 (ICAM-1) in the spinal cord of Lewis rats with actively induced EAE, treated with Rolipram. Treated rats had a delayed and milder disease, and reduced numbers of infiltrates in the nervous tissue. The gene expression profile was similar to that of untreated rats, although delayed, with no evidence of IL-10 upregulation during the observation period. The delayed inflammation was not associated with changes in the expression of VCAM-1 and ICAM-1. In peripheral blood mononuclear cells, TNF mRNA levels were decreased and interleukin-10 was unchanged. This therapy did not alter the proliferative ability of T lymphocytes against myelin basic protein. The encephalitogenic potential of splenocytes from treated animals was also unaffected. The high levels of both iNOs mRNA and nitric oxide (NO) found before the appearance of clinical signs, suggests that NO generation might be a contributing factor to the therapeutic benefit achieved by Rolipram in the rat.
Animals↗
Gene expression analysis of arbuscule development and functioning.
The arbuscular mycorrhiza (AM) is characterized by specific morphological structures of the fungus and the plant and by physiological adaptations which are mostly beneficial for both partners of the symbiosis. This review describes approaches to study the molecular basis of the interaction. RNA accumulation patterns have been monitored in Pisum sativum to analyse the plant response to arbuscule development. In a direct approach, the Mtha1 gene from Medicago truncatula was cloned which is expressed in arbusculated cells of M. truncatula. The gene putatively encodes an H(+)-ATPase involved in the improved plant nutrition during mycorrhization. Finally, a tripartite system between M. truncatula, Glomus mosseae and Aphanomyces euteiches was established, in order to study bioprotection. Analysis of the transcriptome has been started to analyse the interaction between the plant, the pathogen and the AM fungus.
Gene Expression Regulation, Plant↗
Comparing imperfect measurements with the Bland-Altman technique: application in gene expression analysis.
Several problems in medicine and biology involve the comparison of two measurements made on the same set of cases. The problem differs from a calibration problem because no gold standard can be identified. Testing the null hypothesis of no relationship using measures of association is not optimal since the measurements are made on the same cases, and therefore correlation coefficients will tend to be significant. The descriptive Bland-Altman method can be used in exploratory analysis of this problem, allowing the visualization of gross systematic differences between the two sets of measurements. We utilize the method on three sets of matched observations and demonstrate its usefulness in detecting systematic variations between two measurement technologies to assess gene expression.
Bias↗
An adenoviral vector-based system to study neuronal gene expression: analysis of the rat tyrosine hydroxylase promoter in cultured neurons.
We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.
Adenoviridae↗
The use of gene expression analysis to gain insights into signaling mechanisms of metastatic medulloblastoma.
Metastasis is the leading cause of treatment failure in medulloblastoma. Understanding the genetic regulation of metastasis may aid in the development of novel treatments. We therefore performed in silico analysis of the mRNA expression of 83 medulloblastomas compiled from two independent microarray studies by focusing on 135 genes most frequently linked to metastasis in other tumors. We then asked whether expression of these genes correlated with metastasis in the medulloblastoma array data sets. We found the platelet-derived growth factor receptor alpha, early growth response protein 1 and insulin-like growth factor 2 genes as well as several genes associated with MYCC and ERBB2 overexpressed by at least 2-fold in metastatic tumors in both array data sets. We conclude that these genes may interact to promote prometastatic signaling in medulloblastoma.
Cerebellar Neoplasms↗
Gene expression analysis in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease using cDNA microarray: effect of R-apomorphine.
To establish the possible roles of oxidative stress, inflammatory processes and other unknown mechanisms in neurodegeneration, we investigated brain gene alterations in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of Parkinson's disease using Atlas mouse cDNA expression array membrane. The expression of 51 different genes involved in oxidative stress, inflammation, glutamate and neurotrophic factors pathways as well as in still undefined processes, such as cell cycle regulators and signal transduction molecules, was differentially affected by the treatment. The present study indicates the involvement of an additional cascade of events that might act in parallel to oxidative stress and inflammation to converge eventually into a common pathway leading to neurodegeneration. The attenuation of these gene changes by R-apomorphine, an iron chelator-radical scavenger drug, supports our previous findings in vivo where R-apomorphine was neuroprotective.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
Gene expression analysis of largemouth bass exposed to estradiol, nonylphenol, and p,p'-DDE.
The purpose of this study was to determine the specific expression profile of 132 genes, some of which are estrogen responsive, in largemouth bass (LMB) following exposure to estradiol (E(2)), or to two hormonally active agents, 4-nonylphenol (4-NP) and 1,1-dichloro-2, 2-bis (p-chlorophenyl) ethylene (p,p'-DDE), using gene array technology. The results of these experiments show that LMB exposed to E(2) and 4-NP had similar, but not identical genetic signatures for the genes examined, some of which are known to be estrogen-responsive genes. The differences suggest that 4-NP may have additional modes of action that are independent of the estrogen receptor (ER). We have also shown that exposure of male LMB to p,p'-DDE results in an increase in some estrogen-responsive genes. But in female LMB, the observed changes were a down-regulation of the normally up-regulated estrogen responsive genes. Other genes were also down-regulated. These results suggest that p,p'-DDE may affect regulation of genes differently in male and female LMB. This study further suggests that gene arrays have the potential to map out the gene activation pathways of hormonally active compounds.
Animals↗
CDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.
Human cancer is characterized by complex molecular perturbations leading to variable clinical behavior, often even in single-disease entities. We performed a feasibility study systematically comparing large-scale gene expression profiles with clinical features in human B-cell chronic lymphocytic leukemia (B-CLL). cDNA microarrays were employed to determine the expression levels of 1,024 selected genes in 54 peripheral blood lymphocyte samples obtained from patients with B-CLL. Statistical analyses were applied to correlate the expression profiles with a number of clinical parameters including patient survival and disease staging. We were able to identify genes whose expression levels significantly correlated with patient survival and/or with clinical staging. Most of these genes code either for cell adhesion molecules (L-selectin, integrin-beta2) or for factors inducing cell adhesion molecules (IL-1beta, IL-8, EGR1), suggesting that prognosis of this disease may be related to a defect in lymphocyte trafficking. This report demonstrates the feasibility of a systematic integration of large-scale gene expression profiles with clinical data as a general approach for dissecting human diseases.
Adult↗
Gene expression analysis exposes mitochondrial abnormalities in a mouse model of Rett syndrome.
Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked MECP2 gene, which encodes a methyl-CpG binding transcriptional repressor. Using the Mecp2-null mouse (an animal model for RTT) and differential display, we found that mice with neurological symptoms overexpress the nuclear gene for ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1). Chromatin immunoprecipitation demonstrated that MeCP2 interacts with the Uqcrc1 promoter. Uqcrc1 encodes a subunit of mitochondrial respiratory complex III, and isolated mitochondria from the Mecp2-null brain showed elevated respiration rates associated with respiratory complex III and an overall reduction in coupling. A causal link between Uqcrc1 gene overexpression and enhanced complex III activity was established in neuroblastoma cells. Our findings raise the possibility that mitochondrial dysfunction contributes to pathology of the Mecp2-null mouse and may contribute to the long-known resemblance between Rett syndrome and certain mitochondrial disorders.
Animals↗