The role of metabolic cooperation in selection of hypoxanthine-guanine-phosphoribosyl-transferase (HG-PRT)-deficient mutants from diploid mammalian cell strains

Abstract

A system for the selection of 8-azaguanine-resistant mutants from a diploid human cell strain is described. The selection of mutant cells is largely influenced by a phenomenon, known as metabolic cooperation, which turns mutant cells into phenotypically wild-type cells. As a consequence mutant cells cannot be selected above a certain cell density. Reconstruction experiments in which mutant cells were mixed with wild-type cells and mutant feeder cells showed that a reasonable recovery of mutant cells could only be obtained at a density of about 190 wild-type cells per cm 2. Under these conditions HG-PRT (hypoxanthine-guanine-phosphoribosyl-transferase) deficient clones were obtained from a diploid human skin fibroblast strain and from a mouse skin fibroblast strain. In order to improve the selection system the mechanism of metabolic cooperation should be understood. Therefore it was investigated whether metabolic cooperation is due to cell-to-cell contact or to factors mediated by the medium. It is shown that when mutant cells and wild-type cells were separated by a fibrin layer, metabolic cooperation did not occur.