RESUMO
The Escherichia coli xanthine-guanine phosphoribosyltransferase gene (Ecogpt) rescues mammalian cells from inhibition of purine nucleotide biosynthesis by mycophenolic acid (MPA). We used Ecogpt and other selectable markers to obtain subclones of NIH 3T3 derivatives (EN/NIH) stably expressing transfected genes of interest. In their respective selective mediums, growth of MPA-resistant (MPA(R)) isolates was indistinguishable from that of aminoglycoside-resistant counterparts expressing selectable marker genes conferring resistance to protein synthesis inhibitors hygromycin B, puromycin, and G418. Growth of aminoglycoside-resistant isolates remained unaltered on passage to nonselective media. In contrast, MPA(R) cells transferred from MPA complete media to nonselective media displayed morphologic changes with static growth. These findings resolved completely by third passage in nonselective media and were independent of the gene of interest cis-linked to the selectable marker. Sequential selection strategies involving cell culture conditions resulting in these altered growth characteristics significantly impaired detection (by selection in G418) of genomic events associated with reactivation of enhancerless, transcriptionally silent neointegrants present in MPA(R) EN/NIH isolates. We explored the cause of these cell culture findings and defined transfection and sequential selection strategies for MPA(R) derivatives that successfully circumvented these effects.
