We utilized a focus assay on NIH/3T3 cells as a method to isolate genes relevant to growth factor signal transduction. The methodology is based on creating an autocrine transforming loop which results in the growth of a focus that is easily identified and isolated. Genomic DNA from these foci are used to specifically """"""""rescue"""""""" the relevant cDNAs. This protocol was successfully utilized to isolate the keratinocyte growth factor receptor (KGFR). It was known from binding data that a mouse keratinocyte cell line (BALB/MK cells) possessed high affinity binding sites for KGF and that this high affinity receptor was absent from NIH/3T3 cells. NIH/3T3 cells secrete KGF; therefore, the expression of the appropriate cDNA should result in cellular transformation. The introduction of the BALB/MK cDNA expression library into NIH/3T3 cells resulted in the identification of several foci. The KGFR cDNA was """"""""rescued"""""""" from genomic DNA of one of these foci and sequence analysis revealed sequence identity to bek, a member of the fibroblast growth factor receptor (FGFR) gene family. Additionally, NIH/3T3 cells expressing the KGFR cDNA had acquired KGF high affinity binding. This expression cloning strategy should be applicable for isolating other genes critical in growth factor-mediated growth processes.
