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Published on: September 22, 2020
In vivo mutation assay based on the endogenous Pig-a locus
Steven M Bryce1, Jeffrey C Bemis, Stephen D Dertinger
1Litron Laboratories, 200 Canal View Blvd., Rochester, New York 14620, USA.
Environmental and Molecular Mutagenesis
|February 22, 2008
Summary
A novel in vivo mutation assay uses flow cytometry to detect Pig-a gene mutations by identifying cells lacking glycosylphosphatidylinositol (GPI)-anchored proteins. This method successfully identified mutations in rats exposed to mutagens, showing promise for genotoxicity testing.
Area of Science:
- Genetics and Molecular Biology
- Toxicology
- Biochemistry
Background:
- The Pig-a gene is crucial for glycosylphosphatidylinositol (GPI) anchor biosynthesis, essential for cell surface protein attachment.
- A deficiency in GPI-anchored proteins can serve as a biomarker for genetic mutations.
- Developing reliable in vivo mutation assays is vital for toxicological assessments.
Purpose of the Study:
- To evaluate the potential of using the absence of GPI-anchored proteins as a basis for an in vivo mutation assay.
- To establish a flow cytometry method for enumerating cells with Pig-a mutations.
- To assess the sensitivity of the assay in detecting mutagen exposure.
Main Methods:
- Utilized a CD59-negative cell surface phenotype to indicate Pig-a mutation.
- Employed flow cytometry with fluorescent reagents (anti-CD59-PE, thiazole orange, anti-CD61) to analyze erythrocytes, reticulocytes (RETs), and platelets.
- Treated Sprague Dawley rats with model mutagens (ENU, DMBA) and analyzed blood samples post-exposure.
Main Results:
- Reconstruction experiments validated the method's ability to enumerate CD59-negative erythrocytes.
- Mutagen-exposed rats showed significantly elevated frequencies of CD59-negative cells (239-855 x 10(-6) for ENU, 82-405 x 10(-6) for DMBA) compared to controls (18 +/- 19 x 10(-6)).
- Elevated frequencies were generally higher in reticulocytes (RETs) than in total erythrocytes.
Conclusions:
- The study supports the development of an efficient in vivo mutation assay based on flow cytometric detection of aberrant GPI-anchored protein expression in erythrocytes and/or RETs.
- This assay shows promise for identifying genotoxic effects in vivo.
- The CD59-negative phenotype serves as a reliable indicator of Pig-a mutations in this assay.
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01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
01:16In vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.