PASSAGE TO NONSELECTIVE MEDIA TRANSIENTLY ALTERS GROWTH OF MYCOPHENOLIC ACID-RESISTANT MAMMALIAN-CELLS EXPRESSING THE ESCHERICHIA-COLI XANTHINE GUANINE PHOSPHORIBOSYLTRANSFERASE GENE - IMPLICATIONS FOR SEQUENTIAL SELECTION-STRATEGIES

The Escherichia coli xanthine-guanine phosphoribosyltransferase gene ( Ecogpt) rescues mammalian cells from inhibition of purine nucleotide b iosynthesis by mycophenolic acid (MPA), We used Ecogpt and other selec table markers to obtain subclones of MH 3T3 derivatives (EN/NIH) stabl y expressing transfected genes of interest, In their respective select ive mediums, growth of MPA-resistant (MPA(R)) isolates was indistingui shable from that of aminoglycoside-resistant counterparts expressing s electable marker genes conferring resistance to protein synthesis inhi bitors hygromycin B, puromycin, and G418. Growth of aminoglycoside-res istant isolates remained unaltered on passage to nonselective media, I n contrast, MPA(R) cells transferred from MPA complete media to nonsel ective media displayed morphologic changes with static growth, These f indings 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 condit ions resulting in these altered growth characteristics significantly i mpaired detection (by selection in G418) of genomic events associated with reactivation of enhancerless, transcriptionally silent neo integr ants present in MPA(R) EN/NIH isolates, We explored the cause of these cell culture findings and defined transfection and sequential selecti on strategies for MPA(R) derivatives that successfully circumvented th ese effects. (C) 1996 Academic Press, Inc.