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At least 757 records · Page 42Linked to original sources
PubMed2001

[Genomic profiling: from molecular cytogenetics to DNA arrays].

Genetic instability results, in a large majority of solid tumors, in deep chromosomal rearrangements. However, because chromosomal instability produces highly complex caryotypes, rarely showing stereotypic aberrations, it has not been possible to characterize solid cancers according to specific patterns of chromosomal rearrangements. This contrasts with the situation in hematological malignancies, where cytogenetics has allowed to lay out the basis of a renewed classification. New insights have been brought by the development of comparative genomic hybridization (CGH). This molecular cytogenetics approach was originally devised to detect regions in the genome of tumor cells undergoing quantitative changes, i.e. gains or losses of copy numbers. The large body of studies based on CGH show that solid tumors undergo frequent gains and losses and that every chromosomes show at least one region of anomaly. Furthermore, different tumor types present distinct CGH patterns of gains and losses. These observations favor the idea that it may be possible to type human solid cancers according to their patterns of genomic aberrations. However, despite the fact that a number of CGH based studies present data suggesting that different tumor types or cancers at different stages of evolution show distinct patterns of gains and losses, it has proven difficult to be conclusive. This can be mainly attributed to the lack of spatial resolution of CGH. Indeed, CGH uses metaphase chromosomes as hybridization targets and therefore its resolution is at the level of chromosomal banding. The recent adaptation of DNA array technology to CGH will allow to pass this limitation. In DNA array based CGH (array-CGH) metaphase chromosomes have been replaced by spots of cloned DNA. These DNA clones may either be genomic (BACs, YACs or cosmids) or coding (cDNAs). The resolution of array-CGH is therefore determined by the size of the cloned DNA insert (100 Kb for BACs, 1-2 kb for cDNAs). Data corresponding to each of these clones is or will be in a near future linked to DNA sequence data. Hence, in a near future, array-CGH will allow to increase the resolution from a cytogenetic level to a molecular level. Finally, because array technology is highly adaptable to automation, going from classical CGH to array-CGH will produce a quantum leap in throughput.

Chromosome Aberrations↗
PubMed2003

Genome-wide germline-enriched and sex-biased expression profiles in Caenorhabditis elegans.

We performed a genome-wide analysis of gene expression in C. elegans to identify germline- and sex-regulated genes. Using mutants that cause defects in germ cell proliferation or gametogenesis, we identified sets of genes with germline-enriched expression in either hermaphrodites or males, or in both sexes. Additionally, we compared gene expression profiles between males and hermaphrodites lacking germline tissue to define genes with sex-biased expression in terminally differentiated somatic tissues. Cross-referencing hermaphrodite germline and somatic gene sets with in situ hybridization data demonstrates that the vast majority of these genes have appropriate spatial expression patterns. Additionally, we examined gene expression at multiple times during wild-type germline development to define temporal expression profiles for these genes. Sex- and germline-regulated genes have a non-random distribution in the genome, with especially strong biases for and against the X chromosome. Comparison with data from large-scale RNAi screens demonstrates that genes expressed in the oogenic germline display visible phenotypes more frequently than expected.

Animals↗
PubMed2006

Imbalanced genomic imprinting in brain development: an evolutionary basis for the aetiology of autism.

We describe a new hypothesis for the development of autism, that it is driven by imbalances in brain development involving enhanced effects of paternally expressed imprinted genes, deficits of effects from maternally expressed genes, or both. This hypothesis is supported by: (1) the strong genomic-imprinting component to the genetic and developmental mechanisms of autism, Angelman syndrome, Rett syndrome and Turner syndrome; (2) the core behavioural features of autism, such as self-focused behaviour, altered social interactions and language, and enhanced spatial and mechanistic cognition and abilities, and (3) the degree to which relevant brain functions and structures are altered in autism and related disorders. The imprinted brain theory of autism has important implications for understanding the genetic, epigenetic, neurological and cognitive bases of autism, as ultimately due to imbalances in the outcomes of intragenomic conflict between effects of maternally vs. paternally expressed genes.

Autistic Disorder↗
PubMed2002

The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis.

The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways.

Arteriosclerosis↗
PubMed2003

Expression analysis of four Pinus radiata male cone promoters in the heterologous host Arabidopsis.

Four male cone-specific promoters were isolated from the genome of Pinus radiata D. Don, fused to the beta-glucuronidase (GUS) reporter gene and analysed in the heterologous host Arabidopsis thaliana (L.) Heynh. The temporal and spatial activities of the promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1 during seven anther developmental stages are described in detail. The two promoters PrMC2 and PrMALE1 confer an identical GUS expression pattern on Arabidopsis anthers. DNA sequence analysis of the PrMC2 and PrMALE1 promoters revealed an 88% sequence identity over 276 bp and divergence further upstream (<40% sequence identity). GUS expression driven by a 276-bp PrMALE1 promoter fragment showed the same pattern in Arabidopsis anthers as observed for the full-length PrMALE1 promoter. Within the 276-bp promoter fragment a region of high homology to a previously described 16-bp anther-box was identified. In gain-of-function experiments the putative PrMALE1 anther-box was fused upstream of a 90-bp CaMV 35S minimal promoter, as a single copy in the sense direction and as an inverted repeat. No GUS expression was conferred to Arabidopsis anthers by either of these two constructs. In a loss-of-function experiment a 226-bp PrMALE1 deletion construct, which did not contain the putative PrMALE1 anther-box, still maintained the originally observed PrMALE1 GUS expression pattern. Hence, gain-of-function as well as loss-of-function experiments consistently showed that the putative anther-box of the PrMALE1 promoter is non-functional in the Arabidopsis genetic background. For the analysis of the four full-length pine promoters PrCHS1, PrLTP2, PrMC2 and PrMALE1, transformation vectors based on pCAMBIA2200 and pCAMBIA1302 were used. It will also be demonstrated in this article that sequences within the T-DNA borders of these vectors caused a characteristic histological background expression in Arabidopsis, with staining observed in vascular tissue of leaves, sepals, roots, filaments of stamens and in stems and pistils.

Arabidopsis↗
PubMed2004

Latency to onset of status epilepticus determines molecular mechanisms of seizure-induced cell death.

The molecular mechanisms mediating degeneration in response to neuronal insults, including damage evoked by prolonged seizure activity, show substantial variability across laboratories and injury models. Here we investigate the extent to which the proportion of cell death occurring by apoptotic vs. necrotic mechanisms may be shifted by changing the temporal parameters of the insult. In initial studies with continuous seizures (status epilepticus, SE), signs of apoptotic degeneration were most clearly observed when SE occurred following a long latency (>86 min) after injection of kainic acid as compared with a short-latency SE (<76 min). Therefore, in this study we directly compared short- with long-latency SE for the expression of molecular markers for apoptosis and necrosis in an especially vulnerable brain region (rhinal cortex). Molecular markers of apoptosis (DNA fragmentation, cleavage of ICAD, an inhibitor of "caspase-activated DNase" (CAD), and prevalence of a caspase-generated fragment of alpha-spectrin) were detected following long-latency SE. Short-latency SE resulted in expression of predominantly necrotic features of cell death, such as "non-ladder" pattern of genomic DNA degradation, prevalence of a calpain-generated alpha-spectrin fragment, and absence of ICAD cleavage. Silver staining revealed no significant difference in the extent and spatial distribution of degeneration between long- or short-latency SE. These data indicate that the latency to onset of SE determines the extent to which apoptotic or necrotic mechanisms contribute to the degeneration following SE. The presence of a long latency period, during which multiple brief seizure episodes may occur, favors the occurrence of apoptotic cell death. It is possible that the absence of such "preconditioning" period in short-latency SE favors predominantly necrotic profile.

Animals↗
PubMed2005

Coupling cell proliferation and development in plants.

Plant genome projects have revealed that both the cell-cycle components and the overall cell-cycle architecture are highly evolutionarily conserved. In addition to the temporal and spatial regulation of cell-cycle progression in individual cells, multicellularity has imposed extra layers of complexity that impinge on the balance of cell proliferation and growth, differentiation and organogenesis. In contrast to animals, organogenesis in plants is a postembryonic and continuous process. Differentiated plant cells can revert to a pluripotent state, proliferate and transdifferentiate. This unique potential is strikingly illustrated by the ability of certain cells to produce a mass of undifferentiated cells or a fully totipotent embryo, which can regenerate mature plants. Conversely, plant cells are highly resistant to oncogenic transformation. This review discusses the role that cell-cycle regulators may have at the interface between cell division and differentiation, and in the context of the high plasticity of plant cells.

Cell Cycle Proteins↗
PubMed1995

Molecular cloning and pattern of expression of an alpha-L-fucosidase gene from pea seedlings.

alpha-L-Fucosidase is a cell wall protein purified from pea (Pisum sativum) epicotyls. The alpha-L-fucosidase hydrolyzes terminal fucosyl residues from oligosaccharides of plant cell wall xyloglucan. alpha-L-Fucosidase may be an important factor in plant growth regulation, as it inactivates fucose-containing xyloglucan oligosaccharides that inhibit growth of pea stem segments. The amino acid sequences of the NH2-terminal region and one internal peptide were used to design redundant oligonucleotides that were utilized as primers in a polymerase chain reaction (PCR) with cDNA, generated from pea mRNA, as the template. A specific PCR amplification product containing 357 base pairs was isolated, cloned, and sequenced. The deduced amino acid sequence included the two peptides used to design the primers for PCR plus two other peptides obtained by proteinase digestion of alpha-L-fucosidase. No sequence homology to other alpha-L-fucosidases was apparent, although the NH2-terminal region is strongly homologous to Kunitz-type trypsin inhibitors. cDNA and genomic copies were isolated and sequenced. In pea, the gene is present in two or three copies. Its mRNA is present in roots, leaves, and elongating shoots. The spatial pattern of expression of the alpha-L-fucosidase was determined by in situ hybridization.

Amino Acid Sequence↗
PubMed2000

Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.

The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.

Base Sequence↗
PubMed2025

Detection of Orsay viral replication intermediates reveals spatial and regulatory links to Caenorhabditis elegans innate immune responses.

For a positive-strand RNA virus, the encoded viral RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome strand and uses it as a template for amplifying the viral genome, generating various replication intermediates. Structural proteins and viral genome are packaged into virions, but the fate of replication intermediates is underexplored. Here, we investigate Orsay Virus (OV) replication intermediates, including antigenome, oRdRP and double stranded RNA (dsRNA), using PCR and fluorescence-based imaging in C. elegans intestines. As for other positive-strand RNA viruses, we find that genome is in vast excess of antigenome. Antigenome is only visualized in cells when using denaturation protocols, indicating basepaired intermediates. OV antigenome is observed with distinct cytoplasmic and perinuclear localization patterns that depend on factors required for generation of primary, but not secondary, siRNAs. In both wildtype and RNA interference (RNAi) mutants, viral dsRNA is observed in the cytoplasm associated with oRdRP, suggesting cytoplasmic virus replication hubs. Additionally, using antibodies to oRdRP, we observed spherical structures of ~1&#x3bc;m in diameter defined by oRdRP at their surface; over 75% of infected wildtype animals show these structures, which associate with mitochondria and autophagosomes in an antiviral RNAi- and autophagy-dependent manner, respectively. Our study defines new features of OV replication intermediates in wildtype animals, setting the stage for understanding their connection to the viral life cycle and host antiviral pathways.

Journal Article↗
PubMed1992

A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants.

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.

Amino Acid Sequence↗
PubMed2005

Applications of DNA microarrays in biology.

DNA microarrays have enabled biology researchers to conduct large-scale quantitative experiments. This capacity has produced qualitative changes in the breadth of hypotheses that can be explored. In what has become the dominant mode of use, changes in the transcription rate of nearly all the genes in a genome, taking place in a particular tissue or cell type, can be measured in disease states, during development, and in response to intentional experimental perturbations, such as gene disruptions and drug treatments. The response patterns have helped illuminate mechanisms of disease and identify disease subphenotypes, predict disease progression, assign function to previously unannotated genes, group genes into functional pathways, and predict activities of new compounds. Directed at the genome sequence itself, microarrays have been used to identify novel genes, binding sites of transcription factors, changes in DNA copy number, and variations from a baseline sequence, such as in emerging strains of pathogens or complex mutations in disease-causing human genes. They also serve as a general demultiplexing tool to sort spatially the sequence-tagged products of highly parallel reactions performed in solution. A brief review of microarray platform technology options, and of the process steps involved in complete experiment workflows, is included.

Animals↗
PubMed1999

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements.

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.1 4.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.

Bacteria↗
PubMed2001

The spatial and temporal expression of Ch-en, the engrailed gene in the polychaete Chaetopterus, does not support a role in body axis segmentation.

We are interested in understanding whether the annelids and arthropods shared a common segmented ancestor and have approached this question by characterizing the expression pattern of the segment polarity gene engrailed (en) in a basal annelid, the polychaete Chaetopterus. We have isolated an en gene, Ch-en, from a Chaetopterus cDNA library. Genomic Southern blotting suggests that this is the only en class gene in this animal. The predicted protein sequence of the 1.2-kb cDNA clone contains all five domains characteristic of en proteins in other taxa, including the en class homeobox. Whole-mount in situ hybridization reveals that Ch-en is expressed throughout larval life in a complex spatial and temporal pattern. The Ch-en transcript is initially detected in a small number of neurons associated with the apical organ and in the posterior portion of the prototrochophore. At later stages, Ch-en is expressed in distinct patterns in the three segmented body regions (A, B, and C) of Chaetopterus. In all segments, Ch-en is expressed in a small set of segmentally iterated cells in the CNS. In the A region, Ch-en is also expressed in a small group of mesodermal cells at the base of the chaetal sacs. In the B region, Ch-en is initially expressed broadly in the mesoderm that then resolves into one band/segment coincident with morphological segmentation. The mesodermal expression in the B region is located in the anterior region of each segment, as defined by the position of ganglia in the ventral nerve cord, and is involved in the morphogenesis of segment-specific feeding structures late in larval life. We observe banded mesodermal and ectodermal staining in an anterior-posterior sequence in the C region. We do not observe a segment polarity pattern of expression of Ch-en in the ectoderm, as is observed in arthropods.

Amino Acid Sequence↗
PubMed2026

Novel immunotherapeutic strategies for colorectal cancer treatment: Advances, challenges, and future directions.

Immunotherapy has reshaped the treatment landscape of colorectal cancer (CRC), with the clearest and most durable benefit established in mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) disease. However, framing CRC immunotherapy simply as "MSI-H responsive versus microsatellite stable (MSS) resistant" is no longer sufficient. Recent studies indicate that a subset of proficient mismatch repair (pMMR) colon cancers, particularly in the neoadjuvant setting, can mount clinically meaningful responses to immune checkpoint blockade, suggesting that disease stage, local immune organization, and treatment timing critically influence immunotherapy sensitivity. In parallel, emerging evidence has expanded the relevant immune landscape beyond the tumor bed itself, showing that spatially organized stromal and adipose niches can actively divert tumor-reactive lymphocytes and promote immune escape. These advances shift the central challenge in CRC immunotherapy from simply identifying new agents to defining when and in whom immune resistance is reversible, and which biological bottlenecks-such as vascular dysfunction, myeloid suppression, and spatial immune exclusion-must be overcome. In this context, alternative checkpoint inhibitors, bispecific antibodies, cellular therapies, vaccines, nanotechnology-enabled platforms, and microbiome-targeted approaches remain important, but their translational maturity and evidentiary support differ substantially. Biomarker development is likewise evolving from static genomic classification toward dynamic and mechanism-informed stratification incorporating circulating tumor DNA (ctDNA), chromosomal instability, immune architecture, and treatment-induced response trajectories. This review synthesizes recent advances in CRC immunotherapy while emphasizing evidence hierarchy, biomarker-guided patient selection, and the mechanistic basis of combination strategies. We argue that the next phase of CRC immunotherapy will depend less on the indiscriminate addition of novel agents and more on the rational deployment of immunotherapy across molecularly, spatially, and temporally defined disease states.

Humans↗
PubMed1989

Progression to cancer in Barrett's esophagus is associated with genomic instability.

Barrett's esophagus is a condition in which metaplastic columnar epithelium replaces squamous esophageal epithelium as a consequence of chronic gastroesophageal reflux. Patients with this condition are at increased risk for the development of adenocarcinoma. To better understand the progression to adenocarcinoma in this disease, we studied abnormalities in DNA content of epithelial cells in Barrett's esophagus. Using flow cytometry, we examined the spatial distribution of abnormal nuclear DNA contents (aneuploidy) in the esophagi of 14 patients with Barrett's adenocarcinoma. Multiple (2 to 14) populations of aneuploid cells were seen in 12 of the 14 cases. Some early carcinomas appeared to be associated with a single aneuploid population of cells. Surrounding dysplastic epithelium often contained multiple, different overlapping aneuploid populations. These data suggest that neoplastic progression in Barret's esophagus is associated with a process of genomic instability which leads to evolution of multiple aneuploid populations, with the ultimate development of a clone of cells capable of malignant invasion. Thus, detection of multiple aneuploid populations of cells in Barrett's esophagus may indicate a high risk of cancer. Barrett's esophagus provides a unique and readily accessible model for the study of neoplastic progression in human epithelial malignancy.

Adenocarcinoma↗
PubMed2026

FUSE: data-driven functional segmentation of DNA methylation data.

SUMMARY: DNA methylation (DNAm) of neighbouring CpG sites is highly correlated, making DNAm function in terms of blocks. DNAm patterns and functionality are linked to both chromatin structure of DNA and gene regulation. Defining biologically meaningful DNA methylation blocks from whole-genome bisulfite sequencing (WGBS) data remains challenging, as most existing methods rely on fixed genomic windows rather than the observed methylation pattern. We present FUSE, a data-driven segmentation method that captures intrinsic methylation segments directly from WGBS data by jointly analyzing multiple samples. FUSE identifies spatially homogeneous methylation blocks shared across the input cohort while allowing different methylation states across samples. Applied to 61 WGBS samples from the ENCODE database, FUSE identified segments which overlap significantly with promoters, enhancers, and repetitive elements. FUSE was able to recover the true segment breakpoints in synthetic data with high sensitivity under increased levels of noise. As such, FUSE facilitates post hoc methylation analyses by aggregating coherent CpG sites into candidate segments for downstream differential methylation testing or other comparative studies. AVAILABILITY AND IMPLEMENTATION: FUSE is implemented as an R-package methFuse, available at https://github.com/holmsusa/methFuse and https://cran.r-project.org/package=methFuse. A GenomeSpy visualization of the data is available at https://csbi.ltdk.helsinki.fi/p/fuse_encode_gs/.

DNA Methylation↗
PubMed2005

A streamlined method for systematic, high resolution in situ analysis of mRNA distribution in plants.

BACKGROUND: In situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution. RESULTS: Here we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings. CONCLUSION: The approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.

Journal Article↗