TARGETED DISRUPTION OF THE HOUSEKEEPING GENE ENCODING GLUCOSE-6-PHOSPHATE-DEHYDROGENASE (G6PD) - G6PD IS DISPENSABLE FOR PENTOSE SYNTHESIS BUT ESSENTIAL FOR DEFENSE AGAINST OXIDATIVE STRESS

Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene, It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH, In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination, We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable, We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.