Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.
BACKGROUND
A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO A(L)) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 11 but it requires the use of rabbit complement and colony growth for mutant selection. We have developed a more rapid flow cytometry-based mutation assay with CHO A(L) cells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation.


METHODS
CHO A(L) cells were treated with gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then allowed to grow for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry.


RESULTS
Negative and positive populations were separated by over 100-fold. Mixing various proportions of CD59-positive and -negative cells demonstrated that the assay is highly linear (r2 = 0.9999) and sensitive (<0.05% background mutants). The yield of CD59-inducible mutants was linearly related to dose for a clastogen (gamma-radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific.


CONCLUSIONS
Mutations induced by genotoxic agents can be rapidly and sensitively measured in CHO A(L) cells using flow cytometry.
Ross
Carley D
CD
Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523-1618, USA.
Lim
Chang-Uk
CU
Fox
Michael H
MH
D000818:Animals; D000911:Antibodies, Monoclonal; D018957:CD59 Antigens / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D016466:CHO Cells; D002138:Calibration; D002469:Cell Separation; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D006224:Cricetinae; D004252:DNA Mutational Analysis; D005434:Flow Cytometry / Q000379:methods*; D005720:Gamma Rays; D006801:Humans; D006822:Hybrid Cells / Q000187:drug effects / Q000276:immunology / Q000528:radiation effects; D008769:Methylnitronitrosoguanidine; D016296:Mutagenesis / Q000187:drug effects / Q000528:radiation effects; D009152:Mutagenicity Tests / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D018957:CD59 Antigens; D008769:Methylnitronitrosoguanidine