Chapter title |
In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene
|
---|---|
Chapter number | 7 |
Book title |
Drug Safety Evaluation
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7172-5_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7170-1, 978-1-4939-7172-5
|
Authors |
Dobrovolsky, Vasily N., Revollo, Javier, Petibone, Dayton M., Heflich, Robert H., Vasily N. Dobrovolsky, Javier Revollo, Dayton M. Petibone, Robert H. Heflich |
Abstract |
The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Austria | 1 | 33% |
Unknown | 2 | 67% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 3 | 100% |