Mutagenesis at the ouabain-resistance locus in human diploid fibroblasts

Area of Science:

• Cell Biology
• Molecular Toxicology
• Genetics

Background:

• Quantitative mutagenesis studies in human cells require reliable methods for detecting genetic alterations.
• Ouabain-resistance serves as a valuable selectable marker for identifying mutant clones in cell cultures.

Purpose of the Study:

• To determine optimal conditions for the recovery of ouabain-resistant mutant clones in human foetal lung fibroblasts.
• To estimate the spontaneous mutation rate in this cell line.
• To evaluate the efficacy of chemical mutagens and UV radiation in increasing mutant frequencies.

Main Methods:

• Human foetal lung fibroblasts were cultured and treated with varying cell densities and ouabain concentrations for selection.
• Spontaneous and induced mutation rates were assessed following exposure to ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine, and UV light.
• Mutant recovery was optimized by varying cell density and selection concentration, and by introducing post-mutagenesis growth periods.

Main Results:

• Optimal mutant recovery was achieved at a cell density of 2 X 10(4) cells/cm^2 and selection in 10(-6) M ouabain.
• The spontaneous mutation rate was estimated at 4 X 10(-8) per cell generation.
• Mutagenic treatments (EMS, N-methyl-N'-nitro-N-nitrosoguanidine, UV) increased mutant colony frequency by approximately one order of magnitude.

Conclusions:

• Established optimal conditions for quantitative mutagenesis studies using ouabain-resistance in human fibroblasts.
• Demonstrated that ouabain-resistance is a robust genetic marker for assessing mutagenicity in human cells.
• Highlighted the significant increase in mutation frequency achievable with standard mutagens.