The use of correction factors in the determination of mutant frequencies in populations of human diploid skin fibroblasts

Abstract

The need and validity for using a number of correction factors in the determination of mutant frequencies in human diploid skin fibroblasts was investigated. Resistance to the purine analogue 8-azaguanine was used as selective system. It appears that, under the conditions used, there was no effect of the cell density on the cloning efficiency. Therefore observed mutant frequencies need to be corrected for cloning efficiency. The reliability of using Lesch-Nyhan cells as a prototype mutant in the estimation of the efficiency of mutant recovery was investigated and found to hold true. The relation between the efficiency of mutant recovery and cell density, which reflects the degree of inter-clone metabolic cooperation, was measured. The use of this relationship enhances the accuracy of the estimated mutant frequencies. With these correction factors the mutation rate was estimated to be 5.7 · 10 −6 per cell per generation. The influence of intra-clone metabolic cooperation was determined and found to be small if present.