Expression and confirmation of identity of hβc mutants in factor-independent fetal liver cells. (A) Flow cytometric analyses of mutant hβc expression. Dashed lines represent staining with an irrelevant isotype control antibody. Solid lines represent staining with an anti-hβc MoAb. (B) Map of hβc cDNA showing Nco I (N), BstYI (Bs), and Bgl II (Bg) restriction sites used to authenticate each form of hβc, as well as the region duplicated in FIΔ (indicated by boxes). The restriction sites affected by the point mutations are indicated as Bg⁺ (gained in V449E) and Bs⁻ (lost in I374N). Arrows indicate the positions of PCR primers used to amplify hβc fragments from genomic DNA. (C) Electrophoretic analysis of PCR products generated from genomic DNA of factor-independent fetal liver cells infected with constructs containing the indicated hβc mutants. As a negative control, a reaction was performed containing no DNA (−). Lanes M contain DNA size standards (SPP-1 phage DNA digested with EcoRI [Bresatec Ltd, Adelaide, South Australia]). For comparison, PCR products were generated from RufNeo-hβc plasmids (labeled wild-type). PCR products were either undigested (lanes 1), digested with BglII (lanes 2), or digested with BstYI (lanes 3). Bands in each digest that differ between the mutants and the wild-type are indicated by asterisks.