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Results for “comparative transcriptomics”

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PubMed2025

Unveiling the genetic basis of the low pH response in the acidophilic yeast Maudiozyma bulderi as a potential host for biorefinery.

Nonconventional yeasts represent a great genetic and phenotypic diversity with potential for industrial strain development in the bio-production of green chemicals. In recent years, mass genome sequencing of nonconventional yeasts has opened avenues to improved understanding of transcriptional networks and phenotypic plasticity and gene function, including the discovery of novel genes. Here, we investigated the expressional and morphological changes at low-pH in three strains of the acidophilic yeast Maudiozyma bulderi (previously Kazachstania bulderi and Saccharomyces bulderi): CBS 8638, CBS 8639, and NRRL Y-27205. The comparison of the transcriptome of cells growing in a bioreactor at pH = 5.5 vs pH = 2.5, primarily showed dysregulation of genes involved in cell wall integrity, with NRRL Y-27205 the least acidophilic strain, showing the largest transcriptional response when compared to the other strains. We identified four uncharacterized genes, unique to M. bulderi, and predicted function as transporters, upregulated at low pH. Microscopy studies showed that M. bulderi cell wall is not damaged in acidic environment, and the membrane lipid composition remains stable at low pH, unlike Saccharomyces cerevisiae. Overall, our data on transcriptional variability in M. bulderi highlights genes and cellular pathways involved in the acidophilic adaptation of this species and can aid further strain development.

Hydrogen-Ion Concentration↗

PubMed2003

Transcriptomal analysis of failing and nonfailing human hearts.

Heart failure is a multifactorial disease that may result from different initiating events. To contribute to an improved comprehension of normal cardiac function and the molecular events leading to heart failure, we performed large-scale gene expression analysis of failing and nonfailing human ventricle. Our aim was to define and compare expression profiles of 4 specific pathophysiological cardiac situations: 1) left ventricle (LV) from nonfailing heart; 2) LV from failing hearts affected by dilated cardiomyopathy (DCM); 3) LV from failing hearts affected by ischemic CM (ICM); 4) right ventricle (RV) from failing hearts affected by DCM or ICM. We used oligonucleotide arrays representing approximately 12,000 human genes. After stringent numerical analyses using several statistical tests, we identified 1,306 genes with a similar expression profile in all 4 cardiac situations, therefore representative of part of the human cardiac expression profile. A total of 95 genes displayed differential expression between failing and nonfailing heart samples, reflecting a reversal to developmental gene expression, dedifferentiation of failing cardiomyocytes, and involvement of apoptosis. Twenty genes were differentially expressed between failing LV and failing RV, identifying possible candidates for different functioning of both ventricles. Finally, no genes were found to be significantly differentially expressed between failing DCM and failing ICM LV, emphasizing that transcriptomal analysis of explanted hearts results mainly in identification of expression profiles of end-stage heart failure and less in determination of expression profiles of the underlying etiology. Taken together, our data resulted in identification of putative transcriptomal landmarks for normal and disturbed cardiac function.

Adolescent↗

PubMed2006

Under the influence of the active deodorant ingredient 4-hydroxy-3-methoxybenzyl alcohol, the skin bacterium Corynebacterium jeikeium moderately responds with differential gene expression.

A 70mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Corynebacterium jeikeium, a skin bacterium that is predominantly present in the human axilla and involved in axillary odor formation. Oligonucleotides representing 100% of the predicted coding regions of the C. jeikeium K411 genome were designed and spotted in quadruplicate onto epoxy-coated glass slides. The quality of the printed microarray was demonstrated by co-hybridization with fluorescently labeled cDNA probes obtained from exponentially growing C. jeikeium cultures. Accordingly, genes detected with different intensities resulting in log(2) transformed ratios greater than 0.8 or smaller than -0.8 can be regarded as differentially expressed with a confidence level greater than 99%. In an application example, we measured global changes of gene expression during growth of C. jeikeium in the presence of different concentrations of the deodorant component 4-hydroxy-3-methoxybenzyl alcohol that is active in preventing body odor formation. Global expression profiling revealed that low concentrations of 4-hydroxy-3-methoxybenzyl alcohol (0.5 and 2.5mg/ml) had almost no detectable effect on the transcriptome of C. jeikeium. A slightly higher concentration of 4-hydroxy-3-methoxybenzyl alcohol (5mg/ml) resulted in differential expression of 95 genes, 86 of which showed an enhanced expression when compared to a control culture. Besides many genes encoding proteins that apparently participate in transcription and translation, the drug resistance determinant cmx and the predicted virulence factors sapA and sapD showed significantly enhanced expression levels. Differential expression of relevant genes was validated by real-time reverse transcription PCR assays.

Anti-Infective Agents↗

PubMed2026

Genomic and molecular landscape of early onset colorectal cancer: Emerging insights and clinical implications-A systematic review.

BACKGROUND: Early onset colorectal cancer, defined as colorectal malignancy occurring before age 50, has been rising globally. Increasing molecular evidence suggests that early onset colorectal cancer is not merely a premature form of late-onset colorectal cancer but a distinct biologic entity with unique genomic and transcriptomic profiles. METHODS: A systematic PubMed search using the terms "early onset colorectal cancer," "genomic," and "molecular" identified 270 records. Eighteen original studies met the inclusion criteria and were supplemented by references from selected articles. Extracted data encompassed clinicopathologic characteristics, genomic and epigenetic alterations, and dysregulated signaling pathways distinguishing early onset colorectal cancer from late-onset colorectal cancer. RESULTS: Evidence from approximately 19,888 patients with early onset colorectal cancer was synthesized across genomic, transcriptomic, and clinical data sets. Early onset colorectal cancer showed a predominance in distal and rectal sites, a slight male bias, and a higher prevalence among Hispanic and Asian populations. Compared with late-onset colorectal cancer, early onset colorectal cancer exhibited lower B-Raf proto-oncogene, serine/threonine kinase V600E mutation and CpG island methylator phenotype-high methylation frequencies but higher rates of tumor protein p53, Kirsten rat sarcoma viral oncogene homolog, and DNA-repair gene alterations. Distinct comutation patterns (F-box and WD repeat domain containing 7-neurogenic locus notch homolog protein 3-phosphoinositide-3-kinase regulatory subunit 1 and adenomatous polyposis coli-tumor protein p53) and overexpression of immediate-early response genes (Proto-Oncogene c-Fos, EGR1, DUSP1, and CYR61) defined its transcriptional landscape. Perturbations of Wingless/Integrated signaling pathway, mitogen-activated protein kinase, phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin, and DNA-repair pathways, along with global long interspersed nuclear element-1 hypomethylation, indicated heightened genomic instability. CONCLUSION: Early onset colorectal cancer develops through tumor protein p53-driven genomic instability and defective DNA repair rather than the canonical CpG island methylator phenotype-B-Raf proto-oncogene, serine/threonine kinase axis. Recognition of these molecular distinctions is essential for age-specific risk assessment, screening, and precision therapeutics. Further integrative studies are needed to elucidate environmental and genetic contributors and identify novel biomarkers and treatment targets.

Humans↗

PubMed2003

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Adult↗

PubMed2005

A comparative analysis by SAGE of gene expression profiles of Barrett's esophagus, normal squamous esophagus, and gastric cardia.

BACKGROUND & AIMS: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett's esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. METHODS: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. RESULTS: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. CONCLUSIONS: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE.

Adult↗

PubMed2002

Large-scale analysis of the human and mouse transcriptomes.

High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (http://expression.gnf.org) that integrates data visualization and curation of current gene annotations.

Animals↗

PubMed2005

Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture.

Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

Animals↗

PubMed2005

Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes.

BACKGROUND: The extensive use of DNA microarray technology in the characterization of the cell transcriptome is leading to an ever increasing amount of microarray data from cancer studies. Although similar questions for the same type of cancer are addressed in these different studies, a comparative analysis of their results is hampered by the use of heterogeneous microarray platforms and analysis methods. RESULTS: In contrast to a meta-analysis approach where results of different studies are combined on an interpretative level, we investigate here how to directly integrate raw microarray data from different studies for the purpose of supervised classification analysis. We use median rank scores and quantile discretization to derive numerically comparable measures of gene expression from different platforms. These transformed data are then used for training of classifiers based on support vector machines. We apply this approach to six publicly available cancer microarray gene expression data sets, which consist of three pairs of studies, each examining the same type of cancer, i.e. breast cancer, prostate cancer or acute myeloid leukemia. For each pair, one study was performed by means of cDNA microarrays and the other by means of oligonucleotide microarrays. In each pair, high classification accuracies (> 85%) were achieved with training and testing on data instances randomly chosen from both data sets in a cross-validation analysis. To exemplify the potential of this cross-platform classification analysis, we use two leukemia microarray data sets to show that important genes with regard to the biology of leukemia are selected in an integrated analysis, which are missed in either single-set analysis. CONCLUSION: Cross-platform classification of multiple cancer microarray data sets yields discriminative gene expression signatures that are found and validated on a large number of microarray samples, generated by different laboratories and microarray technologies. Predictive models generated by this approach are better validated than those generated on a single data set, while showing high predictive power and improved generalization performance.

Animals↗

PubMed2026

Molecular and immune profiling of HER2-low, HER2 ultra-low, and HER2-null male breast cancer.

BACKGROUND: HER2 expression is described along a biological continuum from null to positive and serves as a critical biomarker for therapeutic guidance in breast cancer (BC). While HER2-low and ultra-low categories have emerged as actionable targets for antibody-drug conjugates (ADCs) in female BC, their molecular and immune characteristics remain largely unexplored in male breast cancer. METHODS: We profiled 214 male breast tumors using next-generation sequencing and whole-transcriptome sequencing to assess mutational, transcriptomic, and immune landscapes. Tumor mutational burden (TMB) was defined as high if > 10 mutations/Mb. Immune cell fractions were inferred using Quantiseq deconvolution. RESULTS: Among 214 samples, 66 (30.8%) were HER2-null, 53 (24.8%) HER2 ultra-low, 80 (37.4%) HER2-low, and 15 (7.0%) HER2-positive. HER2 ultra-low tumors exhibited a higher prevalence of PIK3CA mutations (39.2% vs 22.6%, p ≤ 0.05) compared to HER2-null. No significant differences were observed in TMB-high frequency or PD-L1 expression across subgroups. Immune composition differed primarily between HER2-null and HER2-expressing subgroups: HER2-ultra-low tumors showed higher B-cell infiltration, whereas HER2-null tumors were enriched in neutrophils. Transcriptomic analysis revealed upregulation of selected stemness-associated genes (NANOG, KLF4, POU5F1) and CEACAM1 in HER2-null tumors, while HER2-low and HER2-ultra-low tumors were largely similar across most molecular and immune readouts in this cohort. CONCLUSIONS: HER2-null male breast cancer appears to represent the most biologically divergent subgroup within the HER2-negative spectrum, whereas HER2-low and HER2-ultra-low tumors were largely similar in this cohort. These findings support further investigation of HER2-null disease as a distinct biological state and provide hypothesis-generating data for biomarker development in this rare population.

Male↗

PubMed2006

Evaluating the comparability of gene expression in blood and brain.

The availability of an accessible tissue whose gene expression profile is similar to more inaccessible CNS tissues has the potential to advance research in neuropsychiatric disorders. We conducted secondary data analysis of transcriptional profiling of 79 human tissues for 33,698 genes using the Affymetrix U133A microarray augmented with a custom microarray (Affymetrix GNF1H), which were produced by the Genomics Institute of the Novartis Research Foundation (http://symatlas.gnf.org). Our analyses suggested that: (a) on a transcriptome level, whole blood shares significant gene expression similarities with multiple CNS tissues; (b) the median non-parametric correlation between transcripts present in both whole blood and CNS was around 0.5; (c) this correlation of 0.5 was intermediate relative to all tissues in the Novartis data set--less than for the maximum achievable value of 0.85, less than a set of immune tissues (0.64), comparable to a heterogeneous set of somatic tissues (0.57) but greater than muscle (0.48) and peripheral nervous system tissues (0.36); (d) about half of a set of candidate genes relevant to schizophrenia were expressed in both whole blood and prefrontal cortex; and (e) the expression levels of many classes of biologically relevant processes were not significantly different between whole blood and prefrontal cortex. These analyses suggest that gene expression in whole blood is neither perfectly correlated and useful nor perfectly uncorrelated and useless with gene expression in multiple brain tissues. This suggests that the cautious and thoughtful use of peripheral gene expression may be a useful surrogate for gene expression in the CNS when it has been determined that the relevant gene is expressed in both.

Brain↗

PubMed2005

Microarrays: new tools to unravel parasite transcriptomes.

The ability to monitor the expression levels of thousands of genes in a single microarray experiment is a huge progression from conventional Northern blot analysis or PCR-based techniques. Microarrays can play a pivotal role in the mass screening of genes in a wide range of fields including parasitology. The relatively few parasites that can be readily cultured or isolated from a host, as compared with cell lines or tissue sources, makes microarray technology ideal for maximizing experimental results from a limiting source of starting material. Khan et al. (1999 a) commented in an early review of microarray technology " With this system in place, one can anticipate a time when data from thousands of gene expression experiments will be available for meta-analysis........leading to more robust results and subtle conclusions". Now in 2005, microarrays represent a very powerful resource that can play an important role in the characterization and annotation of the transcriptomes of many parasites of medical and veterinary importance.

Animals↗

PubMed2026

Distinct cell morphotypes of Aureobasidium melanogenum ZN exhibit differential functional profiles in promoting maize growth.

Black yeast-like fungi of the genus Aureobasidium exhibit morphological plasticity, but whether distinct cellular states within the same genetic background are associated with different plant growth-promoting functions remains unclear. Here, yeast-like cells (YL), swollen cells (SC), and chlamydospores (CH) of Aureobasidium melanogenum ZN were characterized. YL was associated mainly with siderophore production and laccase activity, SC with extracellular polysaccharide accumulation, and CH with phosphate mobilization and higher ammonia and IAA production. Whole-genome and comparative genomic analyses revealed a shared repertoire related to nutrient acquisition, auxin-associated metabolism, extracellular oxidation, and carbohydrate remodeling, with expansions in nutrient- and cell-surface-related gene families. Transcriptomic and metabolomic analyses showed distinct deployment of these capacities, with CH exhibiting broad reprogramming of tryptophan-associated, nitrogen, phosphate, central-carbon, and amino-acid metabolism. In maize, CH at the optimal inoculation concentration of 105 CFU·mL-1 produced the strongest growth promotion, increasing plant height, dry biomass, root length, root surface area, and root volume by 58.6%, 365.1%, 191.0%, 194.3%, and 222.4%, respectively. Consistent with this pronounced growth phenotype, maize root transcriptomics showed coordinated CH-induced responses involving root development, nutrient transport, redox regulation, and root-interface remodeling. Root-zone tracking showed greater short-term stability and persistence of CH. These findings identify cellular state as an important functional dimension of Aureobasidium-plant interactions and provide a basis for developing fungal inoculants with defined beneficial cellular states.

Zea mays↗

PubMed2005

Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection.

The Mycobacterium marinum-zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.

Animals↗

PubMed2005

Gene expression profile associated with chronic atrial fibrillation and underlying valvular heart disease in man.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in humans. The pathophysiology of AF involves electrical, structural and contractile remodeling, which is associated with changes in cardiac gene expression. Previous studies of gene-expression changes in clinical AF have mostly been limited to a small number of candidate genes and have not all been well controlled for underlying heart disease. The present study assessed AF-related gene-expression changes in valve-disease patients with microarrays representing the cardiac transcriptome. Right atrial appendages from 11 patients with chronic AF and underlying valvular heart disease (AF-VHD) and seven patients in sinus rhythm with VHD (SR-VHD) were individually compared to an age-matched sinus-rhythm control group (SR-CTRL, 11 patients) using cardiac-specific microarray analysis. One-class statistical analysis was used to identify genes differentially expressed between SR-VHD and SR-CTRL patients. Two-class statistical analysis was used to identify genes differentially expressed between AF-VHD and SR-VHD patients. Out of 3863 analyzed genes, 832 genes were differentially expressed between SR-VHD and SR-CTRL patients, and 169 genes were differentially expressed between AF-VHD and SR-VHD patients. Striking AF-related changes included altered expression of nine genes pointing towards the development of fibrosis (e.g. upregulation of transforming growth factor beta1), and changes in eight genes potentially related to an increased risk of thromboembolic events (e.g. upregulation of alpha2 macroglobulin). Microarray results were confirmed by quantitative PCR. Our results suggest that AF produces a characteristic profile of gene-expression changes that may be related to the pathophysiology of the arrhythmia.

Aged↗

PubMed2026

Blood Bile Acids for Inflammatory Bowel Disease Diagnosis and Disease Activity Assessment: A Metabolomics Meta-Analysis.

Alterations in circulating bile acids (BAs) have been reported in inflammatory bowel disease (IBD), but the consistency of these changes across clinically relevant comparisons remains unclear. Our goal was to investigate systemic BA alterations in IBD using a metabolomics meta-analysis with an exploratory analysis of BA-related gene expression as a supporting context. A systematic review and meta-analysis of 28 metabolomics studies examined blood BA profiles associated with IBD, IBD diagnosis, and disease activity assessment. Univariate analysis and logistic regression modeling of two independent IBD cohorts explored the blood BA-related genes and IBD. Across 28 studies that comprised 5056 IBD patients, 1721 healthy controls, and 314 non-IBD patients, 131 BAs were reported. Eight predefined clinical comparisons were eligible for the meta-analysis. Lower secondary BA levels were consistently observed in IBD patients compared with controls, between UC and CD, and in active versus remission patients. Deoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid were frequently decreased, whereas glycocholic acid was increased in certain comparisons. Transcriptomics analyses revealed differential expression of several BA-related genes in blood, including SLC51A, ABCB4, and ACOT8, across the comparisons. Our findings identify consistent circulating BA alterations in IBD and highlight the relevance of blood BA for future biomarker research in the diagnosis and disease activity assessment.

Humans↗

PubMed2006

DeepSAGE--digital transcriptomics with high sensitivity, simple experimental protocol and multiplexing of samples.

Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.

Diphosphates↗

PubMed2004

Global gene expression analysis comparing bovine blastocysts flushed on day 7 or produced in vitro.

In vitro produced (IVP) bovine embryos have darker cytoplasm, reduced buoyant density, fragile zonae pellucidae, chromosomal abnormalities, higher pregnancy failure rates, and altered gene expression compared to embryos produced in vivo. Characterization of early deviations in gene expression would enable us to better understand the biology of early embryo development and improve in vitro culture systems. Here we compared gene expression between Day 7 blastocysts generated in TCM199 with 5% FBS and Day 7 in vivo derived blastocysts and using suppression-subtractive hybridization (SSH). Pools of 25 embryos for both driver and tester were used in the RNA extraction process. The subtracted products were cloned and subjected to differential hybridization screening analysis. cDNAs were isolated, single-pass sequenced, and subjected to BLAST search. Of 32 in vivo ESTs (expressed sequence tags) that provided sequence information, 30 matched homologous sequences in GenBank. Of 32 in vitro ESTs, 22 provided specific matches while the remaining ten represented novel transcripts. Two in vivo ESTs, galectin-1 and fibronectin, and one in vitro EST, filamin A, were further characterized using real-time quantitative PCR. To further examine the reproducibility of the SSH data, three different pools of embryos with each pool containing ten embryos produced from each of the following production systems, namely, in vivo, IVP in TCM199 with 5% FBS and CR1aa with 5% FBS were used for real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmation studies. Significant increases in the expression level of galectin-1 and fibronectin were observed in the in vivo derived blastocysts compared to blastocysts produced in TCM199 with 5% FBS and CR1aa cultures. No significant difference in filamin A expression was found between blastocysts produced in vivo and those derived from either of the in vitro production systems. We conclude that these techniques are useful to characterize the transcriptome of the early preattachment embryo and observed deviations in mRNA expression may partially explain the differences in quality between in vivo and IVP embryos.

Animals↗