However, within two days of growth factor removal, the vast majority of cells expressing RFP died, whereas the cells expressing TC2-3 and GFP proliferated due to activation of the hEPOR by TC2-3. The relative proportion of GFP + cells in the population increased with extended incubation times in the absence of growth factors. Thus, cells expressing TC2-3 do not secrete a factor that stimulates growth of BaF3/ hEPOR cells lacking TC2-3 in the same culture, demonstrating that TC2-3 activates the hEPOR in a cell-autonomous manner. Because of this property, we were able to use a genetic method to screen a large number of TC2-3 mutants in mixed culture for those with increased activity, because the effect of each mutant is restricted to the cell expressing it, thereby allowing us to isolate rare active clones. We also needed a system in which TC2-3 was minimally active, so that a more active version would confer a selectable phenotype. BaF3/hEPOR cells grow robustly in the absence of growth factors if TC2-3 was expressed from a high expression vector, such as T2H-F13, but low-level expression of TC2-3 from the RVY-hygro vector supports minimal growth factor-independent proliferation. Thus, TC2-3 mutants that induced growth factor independence when expressed at a low level in BaF3/hEPOR cells were likely to be more active than TC2-3 itself. To isolate TC2-3 mutants with enhanced activity, we subjected the transmembrane domain of TC2-3 to limited Cantharidin random mutagenesis. We used a degenerate oligonucleotide in which each position encoding the transmembrane segment was synthesized with a nucleotide mixture consisting of the wild-type nucleotide ����doped���� with a low percentage of each non-wild-type nucleotide. This oligonucleotide was converted into double-stranded DNA, Diosmetin amplified, and cloned into the low expression vector, pRVY-puro, to generate a library named TC2-3.LRM, which encodes an estimated 15,000 different TC2-3 mutants with an average of two to three amino acid substitutions per protein. Figure 1D shows the strategy used to isolate mutants of TC2-3 with increased activity.