FIG. 2.

Double drug selection with TK and Zeo under control of the E-selectin promoter. (A) E-selectin–TK and E-selectin–Zeo. An upstream fragment of the E-selectin gene (−730 to +52), containing one ATF and three NF-κB binding sites and a TATA box, was cloned in front of the TK cDNA or the Zeo gene. (B) Drug selection. E-selectin–TK and E-selectin–Zeo were cotransfected into 293 cells, and the transfected cells were selected in Zeo plus IL-1. Individual clones were assayed for survival in gancyclovir (GCV), death in gancyclovir plus IL-1, death in Zeo, and survival in Zeo plus IL-1. One such clone was expanded and subjected to five rounds of mutagenesis. IL-1-unresponsive mutants were isolated by selecting the mutagenized pools in gancyclovir plus IL-1. Putative mutants were then tested for survival in gancyclovir and gancyclovir plus IL-1 and for death in Zeo and Zeo plus IL-1.