A method for the isolation of purine nucleoside phosphorylase-deficient variants of mammalian cell lines

A method is described for the isolation of purine nucleoside phosphorylase (PNP) deficient lines of both Novikoff hepatoma and CHO cells. This method utilizes the ability of these cell lines to grow on inosine as a carbon and energy source. Using BrdU-light selection, inosine-negative survivors appeared at a frequency of about 10^–5 and PNP-deficient cells appeared at a frequency of about 10^–6. The PNP-negative cell line isolated from Novikoff hepatoma cells, ino-6, was temperature sensitive for both growth and PNP activity. The low activity seen after growth at 37° was approximately 5% of the wild-type level and had a tenfold increase in the apparent K_m for inosine. The extracts of the variant contained a protein that cross-reacted with antiserum made against purified rat liver PNP. This method should be useful for the isolation of PNP-deficient cells in any cell line capable of utilizing inosine for growth.