Abstract:

This is a laboratory-based project to identify the mutagenic capacity of oil compounds. The Hepa1c1c2 cell line is used and the endogenous Piga gene is the target DNA for mutation assessment. 5 x 10^5 Hepa1c1c7 cells were plated in 60 mm dishes and allowed to attach overnight. The following morning cells were treated with Dimethyl sulfoxide (DMSO) solvent controls (<0.1% by volume) and DMSO-Polycyclic aromatic hydrocarbon (PAH) solutions with final concentrations of 0.3 and 3.0 micromolar for 6 hours in serum-free medium. Cells were washed and expanded in culture for a minimum of 8 days for expression of the mutant phenotype. Following mutant expression 10^6 – 20^6 cells were collected and incubated with a PE-conjugated primary antibody for a GPI linked cell surface marker (CD-59). These cell populations were then immunomagnetically enriched for CD_59 negative cells by passing them through a magnetic cell separator (miltenyi LD column) following conjugation with an anti-PE paramagnetic bead. The mutant-enriched cell populations were evaluated with flow cytometry and increases in CD-59 negative cell populations were taken to represent chemically induced Piga gene mutants resulting in a CD59 phenotype. The mutagenic potency of the PAHs (Phenanthrene, Chrysene, 5,12-dimethyl-Chrysene, Dibenz[a,h]anthracene, Benzo[a]anthracene, and Benzo[k]fluoranthene) were evaluated by using Benzo[a]Pyrene as an index chemical.

Suggested Citation:

Wilson, Mark. 2020. Piga gene mutation assay. Distributed by: Gulf of Mexico Research Initiative Information and Data Cooperative (GRIIDC), Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/WJF27X6M

Purpose:

Optimize in vitro mutation assay method. Test PAHs for differential toxicity.

Data Parameters and Units:

The piga gene mutation assay folder contains the multiple subfolders with dates: 3/6/2019, 5/10/2019, 7/24/2019, 7/31/2019, 8/2/2019, 8/16/2019, 8/20/2019, 10/7/2019. Each of these sub-folders contains flow cytometry data files (file extension .mqd) generated on a Miltenyi MacsQuant 10 multiparameter flow cytometer. Miltenyi MACSQuantify software is required to read the files. Each folder contains an additional subfolder labeled analysis. Within the analysis folder, there is a sequential gating and mutant event enumeration file (with extension .glf). These files were generated with Flowlogic Software version 700.2A, which is required in order to read the files. Please note the Excel file “~$BaP passage vs all cells.xlsx” in the folder "piga gene mutation assay/2019-03-06/Analysis” is not a part of the dataset.

Instruments:

MacsQuant 10 flow cytometer (Miltenyi) Flow Logic software (version 700.2a)

Individual Files: