scholarly journals Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.

1990 ◽  
Vol 38 (7) ◽  
pp. 917-922 ◽  
Author(s):  
S Ozden ◽  
C Aubert ◽  
D Gonzalez-Dunia ◽  
M Brahic
Keyword(s):  
In Situ Hybridization ◽  
Theiler's Virus ◽  
Bhk Cells ◽  
Beta Actin ◽  
Actin Mrna ◽  
High Level ◽  
Radiolabeled Probe ◽  
In Situ Detection

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat

1998 ◽  
Vol 111 (9) ◽  
pp. 1287-1292 ◽  
Author(s):  
H. Watanabe ◽  
E.H. Kislauskis ◽  
C.A. Mackay ◽  
A. Mason-Savas ◽  
S.C. Marks
Keyword(s):  
In Situ Hybridization ◽  
Bone Resorption ◽  
Cell Types ◽  
Mrna Isoforms ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Processes ◽  
Actin Mrna ◽  
Actin Isoform

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

scholarly journals Apoptotic Cells, Including Macrophages, Are Prominent in Theiler's Virus-Induced Inflammatory, Demyelinating Lesions

2003 ◽  
Vol 77 (7) ◽  
pp. 4383-4388 ◽  
Author(s):  
Brian P. Schlitt ◽  
Matthew Felrice ◽  
Mary Lou Jelachich ◽  
Howard L. Lipton
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Apoptotic Cells ◽  
Theiler's Virus ◽  
Demyelinating Lesions ◽  
Encephalomyelitis Virus ◽  
Mouse Central Nervous System

ABSTRACT Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system principally in macrophages, and infected macrophages in culture undergo apoptosis. We have detected abundant apoptotic cells in perivascular cuffs and inflammatory, demyelinating lesions of SJL mice chronically infected with TMEV. T cells comprised 74% of apoptotic cells, while 8% were macrophages, 0.6% were astrocytes, and ∼17% remained unidentified. In situ hybridization revealed viral RNA in ∼1% of apoptotic cells.

scholarly journals Human neutrophilic and eosinophilic granulocytes display different levels of c-fos proto-oncogene expression: an in situ hybridization study.

1987 ◽  
Vol 35 (8) ◽  
pp. 837-842 ◽  
Author(s):  
H Kreipe ◽  
H J Radzun ◽  
K Heidorn ◽  
C Mäder ◽  
M R Parwaresch
Keyword(s):  
In Situ Hybridization ◽  
Neutrophilic Granulocytes ◽  
Blood Monocytes ◽  
In Vitro Differentiation ◽  
Monocytic Differentiation ◽  
Eosinophilic Granulocytes ◽  
Fos Expression ◽  
High Level

The cellular homologue of the retroviral oncogene v-fos has been shown to be involved in cell differentiation of hematopoietic cells. By use of the human promyelocyte cell line HL-60, several in vitro differentiation studies suggested a selective activation of c-fos during monocytic differentiation of myeloid precursor cells. In contrast to these observations, we found high levels of c-fos mRNA in purified normal human granulocytes, whereas c-fos was only faintly expressed in blood monocytes. In situ hybridization revealed that the high level of c-fos expression is restricted to neutrophilic granulocytes, whereas c-fos transcription is not detectable in eosinophilic granulocytes. These results indicate that in vitro differentiation systems can be misleading and may not reflect the in vivo situation. The high level of c-fos expression in neutrophilic granulocytes may be caused by superinduction due to the reduced capacity for protein synthesis in these cells.

scholarly journals The chicken c-erbA proto-oncogene is preferentially expressed in erythrocytic cells during late stages of differentiation.

1987 ◽  
Vol 7 (7) ◽  
pp. 2416-2424 ◽  
Author(s):  
D Hentzen ◽  
A Renucci ◽  
D le Guellec ◽  
M Benchaibi ◽  
P Jurdic ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Life Span ◽  
Blood Cells ◽  
Low Levels ◽  
High Level ◽  
Nuclease Mapping ◽  
Kidney Liver ◽  
Late Stages ◽  
Different Tissues

We analyzed the expression of the c-erbA proto-oncogene in different tissues of chicken embryos. c-erbA transcripts were found at low levels in the lung, kidney, liver, and heart and in high amounts in embryonic blood cells. Nuclease mapping assays proved that these transcripts were true c-erbA transcripts. In situ hybridization on fractionated embryonic blood cells showed that c-erbA transcripts were predominantly found in erythroblasts, particularly during the final step of differentiation. Life span analysis of c-erbA mRNAs revealed their relative instability, demonstrating that the high level of c-erbA transcripts in embryonic erythroblasts was not the result of passive accumulation. These results suggest that the c-erbA genes play some role in erythrocyte differentiation.

Comparison of detection specificity of nitrifying bacteria in biofilm using fluorescence in situ hybridization and in situ fluorescent antibody methods

2003 ◽  
Vol 47 (5) ◽  
pp. 129-132
Author(s):  
N. Noda ◽  
Y. Ebie ◽  
M. Matsumura ◽  
S. Tsuneda ◽  
A. Hirata ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Specific Fluorescence ◽  
Antibody Method ◽  
In Situ Detection ◽  
Detection Specificity ◽  
Fluorescent Antibody Method

The in situ fluorescent antibody and fluorescence in situ hybridization (FISH) methods are very useful in the in situ detection of specific bacteria like nitrifiers in a biofilm. In this study, simultaneous staining using the FISH and in situ fluorescent antibody methods was examined. As a result, no specific fluorescence was observed with either method when FISH was performed followed by the in situ fluorescent antibody method; however, when the in situ fluorescent antibody method was performed first followed by FISH, specific fluorescence was observed in both cases. Moreover, it was suggested that the detection specificities of FISH and the in situ fluorescent antibody method are almost identical.

scholarly journals HuluFISH non-denaturing in situ detection of genomic DNA opened by CRISPR-Cas9 Nickase and Exonuclease

2021 ◽  
Author(s):  
Kang Han ◽  
Sheng Liu ◽  
Yongsheng Cheng
Keyword(s):  
In Situ Hybridization ◽  
Genomic Dna ◽  
Genetic Research ◽  
Artificial Chromosome ◽  
Heat Denaturation ◽  
Novel Method ◽  
Exonuclease Digestion ◽  
In Situ Detection ◽  
Novel Design

DNA fluorescence in situ hybridization (FISH) has been widely used in diagnosis and genetic research. Traditional Bacterial artificial chromosome (BAC) or oligonucleotide based probe to detect DNA in situ is only effective when the target is relatively large, usually over 150Kb DNA fragments. And it involves heat denaturation steps to open the DNA for in situ hybridization. The heat process can affect the fine structure of nuclei. Here we reported a novel method based on Cas9 nickase and exonuclease digestion of double strand DNA and permanently mark the DNA in single strand state for FISH. With this novel design, we detected non-repetitive genomic loci as small as 2Kb.

scholarly journals In Situ Detection of an Uncultured Predominant Bacillus in Dutch Grassland Soils

1998 ◽  
Vol 64 (11) ◽  
pp. 4588-4590 ◽  
Author(s):  
Andreas Felske ◽  
Antoon D. L. Akkermans ◽  
Willem M. De Vos
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Cell Type ◽  
Grassland Soils ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Whole Cell ◽  
Shaped Cell ◽  
In Situ Detection

ABSTRACT Uncultured predominant Bacillus ribotype DA001 in Dutch Drentse A grassland soils, as revealed by its 16S rRNA sequence, was detected in soil by fluorescent whole-cell in situ hybridization. A prominent rod-shaped cell type was identified in bacterial suspensions prepared from soil by a multiple 16S rRNA probing approach.

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.

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

1989 ◽  
Vol 258 (2) ◽  
Author(s):  
Shigeru Sakiyama ◽  
Yohko Nakamura ◽  
Katsuo Tokunaga ◽  
Hiroshi Takazawa ◽  
Yoshinori Ohwaki ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Seminiferous Tubules ◽  
Intestinal Epithelia ◽  
Specific Localization ◽  
Actin Mrna

scholarly journals The localization of actin mRNA in megakaryocytes demonstrated by in situ hybridization using digoxigenin-labeled riboprobes.

1991 ◽  
Vol 24 (3) ◽  
pp. 335-339
Author(s):  
JUNZO SASAKI ◽  
TAKAKO NOMURA ◽  
SADAHIRO WATANABE ◽  
SHIGETO KANDA ◽  
TOSHIYUKI KOUNO ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Actin Mrna
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