Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry.
We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A(L)) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A(L) cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis.
Ross
Carley D
CD
Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523, USA.
French
C Tenley
CT
Keysar
Stephen B
SB
Fox
Michael H
MH
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D018957:CD59 Antigens / Q000235:genetics; D016466:CHO Cells; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D027261:Fusion Regulatory Protein-1 / Q000235:genetics; D015870:Gene Expression; D005819:Genetic Markers; D017261:Glycosylphosphatidylinositols / Q000235:genetics*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics; D006822:Hybrid Cells; D009154:Mutation; D016133:Polymerase Chain Reaction; D060185:Tetraspanin 24; D018800:Thy-1 Antigens / Q000235:genetics
D015703:Antigens, CD; C554715:CD151 protein, human; C497483:CD44 protein, human; D018957:CD59 Antigens; D027261:Fusion Regulatory Protein-1; D005819:Genetic Markers; D017261:Glycosylphosphatidylinositols; D018960:Hyaluronan Receptors; D060185:Tetraspanin 24; D018800:Thy-1 Antigens; C051590:CD59 protein, human