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Isolation of micronuclei from mouse blood and fluorescence in situ hybridization with a mouse centromeric DNA probe
M Hayashi1, J Mäki-Paakkanen, H Tanabe
1Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan.
Abstract:
Spontaneously existing and chemically induced micronuclei were isolated from mouse blood. 50 microliters of cardiac blood was diluted with PBS and centrifuged. After this, the cell pellet was subjected to hypotonic treatment, fixed with acetic acid-methanol (1:3), and the lysate was filtrated through a 2-microns polycarbonate nucleopore membrane. Isolated micronuclei were air-dried on a glass slide and subjected to fluorescence in situ hybridization (FISH) using a mouse centromeric gamma satellite probe. Approximately half of the micronuclei isolated from vehicle control mice showed centromere signal(s). In these preliminary studies, the proportion of centromere-positive micronuclei was increased by treatment with spindle poisons (colchicine and vinblastine sulfate), decreased only slightly by 1-beta-D-arabinofuranosylcytosine, and was generally unaffected by mitomycin C.
Insights
This study isolated micronuclei from mouse blood to analyze their centromeric content. Chemical treatments affecting spindle fibers significantly increased centromere-positive micronuclei, indicating their utility in genotoxicity testing.
Area of Science:
- Cytogenetics
- Genotoxicology
- Molecular Biology
Background:
- Micronuclei are biomarkers of chromosomal instability and genotoxicity.
- Centromere detection in micronuclei helps distinguish aneugenic from clastogenic events.
- Accurate isolation and detection methods are crucial for reliable genotoxicity assessment.
Purpose of the Study:
- To develop and validate a method for isolating and analyzing centromere-positive micronuclei from mouse blood.
- To investigate the effect of various chemical agents on the proportion of centromere-positive micronuclei.
Main Methods:
- Isolation of micronuclei from mouse blood via hypotonic treatment, fixation, and filtration.
- Fluorescence in situ hybridization (FISH) using a mouse centromeric gamma satellite probe.
- Analysis of centromere signals in micronuclei from control and chemically treated mice.
Main Results:
- A method for isolating and analyzing micronuclei with centromere signals was established.
- Approximately 50% of micronuclei in control mice exhibited centromere signals.
- Spindle poisons (colchicine, vinblastine sulfate) significantly increased centromere-positive micronuclei.
- 1-beta-D-arabinofuranosylcytosine showed a slight decrease, while mitomycin C had no significant effect.
Conclusions:
- The developed method effectively identifies centromere-positive micronuclei in mouse blood.
- The results support the use of centromere-positive micronuclei analysis for distinguishing aneugenic effects of chemicals.
- This approach offers a valuable tool for genotoxicity testing and understanding mechanisms of chromosomal damage.