Tissue- and Site-Specific Gene Expression of Type 2 17 -Hydroxysteroid Dehydrogenase: In Situ Hybridization and Specific Enzymatic Activity Studies in Human Placental Endothelial Cells of the Arterial System

2000 ◽  
Vol 85 (12) ◽  
pp. 4841-4850 ◽  
Author(s):  
M. Bonenfant
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Hydroxysteroid Dehydrogenase ◽  
Arterial System ◽  
Specific Gene ◽  
Site Specific ◽  
Specific Enzymatic Activity

scholarly journals Gene expression analysis by non-radioactive RNA-RNA in situ hybridization techniques

2004 ◽  
Vol 23 (2) ◽  
pp. 127-133 ◽  
Author(s):  
Danijela Drakulic ◽  
Milena Stevanovic ◽  
Gordana Nikcevic
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cultured Cells ◽  
Specific Gene ◽  
Rna In Situ Hybridization ◽  
Sox Gene ◽  
Labeled Rna ◽  
Set Up

RNA-RNA in situ hybridization is a reliable method for studying tissue and cell specific gene expression, which enables visualization of labeled antisense RNA probe hybridized to specific mRNA. In this study we employed non-radioactive RNA-RNA in situ hybridization using biotin- or digoxigenin-labeled RNA probes in order to detect SOX gene expression in carcinoma cell lines. By this approach we confirmed results obtained by Northern blot analysis, where the presence of SOX2 mRNA in NT2/D1 and SOX14 mRNA in HepG2 cells has been established. Our aim was to set up RNA-RNA in situ hybridization method in in vitro cultured cells in order to perform further analyses of SOX gene expression on various normal and cancer tissues.

scholarly journals Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

1989 ◽  
Vol 37 (5) ◽  
pp. 697-701 ◽  
Author(s):  
F J Tang ◽  
P O Ts'o ◽  
S A Lesko
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Specific Gene ◽  
Whole Cells ◽  
Beta Interferon ◽  
Ht1080 Cells ◽  
In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

Presence and distribution of endothelin-1 gene expression in human kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F679-F687 ◽  
Author(s):  
C. Pupilli ◽  
M. Brunori ◽  
N. Misciglia ◽  
C. Selli ◽  
L. Ianni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Renal Cortex ◽  
Cdna Probe ◽  
Renal Medulla ◽  
Human Kidney ◽  
Renal Tumors ◽  
Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

Gene expression profiling of microdissected Hodgkin Reed-Sternberg cells correlates with treatment outcome in classical Hodgkin lymphoma

Blood ◽  
2012 ◽  
Vol 120 (17) ◽  
pp. 3530-3540 ◽  
Author(s):  
Christian Steidl ◽  
Arjan Diepstra ◽  
Tang Lee ◽  
Fong Chun Chan ◽  
Pedro Farinha ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Treatment Failure ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Specific Gene ◽  
Classical Hodgkin Lymphoma

Abstract In classical Hodgkin lymphoma (CHL), 20%-30% of patients experience relapse or progressive disease after initial treatment. The pathogenesis and biology of treatment failure are still poorly understood, in part because the molecular phenotype of the rare malignant Hodgkin Reed-Sternberg (HRS) cells is difficult to study. Here we examined microdissected HRS cells from 29 CHL patients and 5 CHL-derived cell lines by gene expression profiling. We found significant overlap of HL-specific gene expression in primary HRS cells and HL cell lines, but also differences, including surface receptor signaling pathways. Using integrative analysis tools, we identified target genes with expression levels that significantly correlated with genomic copy-number changes in primary HRS cells. Furthermore, we found a macrophage-like signature in HRS cells that significantly correlated with treatment failure. CSF1R is a representative of this signature, and its expression was significantly associated with progression-free and overall survival in an independent set of 132 patients assessed by mRNA in situ hybridization. A combined score of CSF1R in situ hybridization and CD68 immunohistochemistry was an independent predictor for progression-free survival in multivariate analysis. In summary, our data reveal novel insights into the pathobiology of treatment failure and suggest CSF1R as a drug target of at-risk CHL.

scholarly journals Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2678
Author(s):  
Judit Vágó ◽  
Katalin Kiss ◽  
Edina Karanyicz ◽  
Roland Takács ◽  
Csaba Matta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
In Situ Hybridization ◽  
Marker Genes ◽  
Specific Gene ◽  
Cartilage Formation ◽  
Methylation Inhibitor ◽  
Dna Methylation Inhibitor

We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

Use of fluorescence in situ hybridization to detect Felis catus papillomavirus type 2 in feline Bowenoid in situ carcinomas

2018 ◽  
Vol 21 (6) ◽  
pp. 575-580 ◽  
Author(s):  
Lauren E Demos ◽  
John S Munday ◽  
Christian E Lange ◽  
Mark D Bennett
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Squamous Cell ◽  
Squamous Cell Carcinomas ◽  
Felis Catus ◽  
Pcr Detection ◽  
Lesion Formation ◽  
Site Specific

Objectives Papillomaviruses (PVs) are ubiquitous host- and site-specific viruses. PV infections in cats are associated with oral papillomas, viral plaques, Bowenoid in situ carcinomas (BISCs), squamous cell carcinomas and sarcoids; this association is primarily based on PCR detection of PV DNA within said lesions. PV DNA is frequently detectable on normal feline skin; thus, it is possible that some of the implicated DNA is commensal rather than associated with lesion formation. Therefore, the aim of the present study was to use fluorescence in situ hybridization (FISH) to localize PV DNA within feline BISCs, to provide additional evidence that PV infection may influence the development of these neoplasms. Methods FISH probes targeting Felis catus papillomavirus type 2 (FcaPV2) DNA were used to localize FcaPV2 DNA within 42 BISCs from which FcaPV2 DNA had previously been amplified via PCR. Results Fifteen of 42 BISC lesions (35.7%) demonstrated intralesional FcaPV2 using FISH. Probe annealing was predominantly located within the nuclei of koilocytes found in the upper strata of the epidermis. Probes were typically scattered multifocally within the lesions; most commonly this was near the periphery of the BISCs. Conclusions and relevance These results confirm that a proportion of BISCs contain FcaPV2 DNA. These results further support a causative association between FcaPV2 and BISCs in cats.

Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance

1992 ◽  
Vol 262 (1) ◽  
pp. G123-G130 ◽  
Author(s):  
P. G. Traber ◽  
L. Yu ◽  
G. D. Wu ◽  
T. A. Judge
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Stem Cell Population ◽  
Specific Gene ◽  
Mrna Levels ◽  
Human Small Intestine ◽  
Crypt Cells

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

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