Fig. 1

Cloning of cDNAs that mediate toxin resistance

MCF-7/T cells expressing SV40 T antigen were obtained from MCF-7 cells by cotransfection of pCMV-TAg (23) and pMC1neo polyA (Stratagene) and subsequent G418 selection at 0.8 mg/ml as described in Materials and Methods. MCF-7 and MCF-7/T cells have the same morphology and sensitivity to PE and B3-derived immunotoxins but MCF-7/T appears to grow slightly slower. MCF-7/T were transfected with a pCDM8/HeLa cDNA expression library, treated with 10 ng/ml immunotoxin (B3(Fv)-PE38KDEL) (9); and three days later the flasks were washed with PBS, and the remaining cells were trypsinized, plasmids reisolated from them (25) and transformed into E. coli MC1061/P3. Aliquots of the transformations were used to determine the number of recovered plasmids (cfu) in E. coli, the remainder was grown for preparation of plasmid pools to be again transfected into MCF-7/T for more rounds of selection. After the third round of transfection-selection, stable MCF-7 transfectants were made by cotransfection of single isolated plasmids and pMC1neo/polyA.