We infected several pools of BaF3/HAhEPOR cells with the TC2-3.LRM library at a low multiplicity of infection, selected with puromycin for stable transduction of the mutant TC2-3 genes, and incubated transduced cells in the absence of growth factors. After eight days, cells infected with the library proliferated robustly in the absence of growth factors, but cells infected with the empty RVY-puro vector did not. The library inserts from the genomic DNA of these growth factorindependent cells were amplified,Songorine cloned as pools into pRVYpuro, and packaged en masse to generate individual secondary libraries. After infecting naı ¨ve BaF3/HA-hEPOR cells with each secondary library and repeating the selection for growth factor independence, a number of TC2-3 mutants were recovered from proliferating cells. Each of these mutants contains a single amino acid substitution at a different position in the mutagenized transmembrane segment. These mutants were expressed individually from RVY-puro in BaF3/HA-hEPOR cells and tested for their ability to confer growth factor independence. Several of these TC2-3 mutants were more active than TC2-3 in this assay. Immunoprecipitation and Western blotting revealed that most of these TC2-3 mutants were not expressed at higher levels than TC2-3 itself, so their increased activity is not simply a consequence of increased expression. One mutant,Benzoylmesaconine designated EBC5-16, contains an isoleucine to serine mutation at position 25 and was reproducibly the most active in conferring growth factor independence. Inserting any of the other mutations identified in the screen into EBC5-16 did not further enhance its activity, so we focused on EBC5-16 itself for further experiments. For comparisons between TC2-3 and EBC5-16 and between the Put3/EBC5-16 chimeras, we typically used the RVY low expression vector. In most other experiments, we used the higher expression vector MSCV to obtain more robust activity. When EBC5-16 was expressed from the high expression vector, MSCV, it did not confer growth factor independence in parental BaF3 cells lacking hEPOR expression, and like TC2-3 itself displayed minimal activity in BaF3 cells expressing the murine EPOR or PDGFbR.