Generation and characterization of novel C57BL/6-derived TF cell lines. (A) Schematic representation of the wild-type TF expression vector used to generate TF^WT cells. The TF expression vector was generated by cloning a PCR-amplified murine TF cDNA into the multiple cloning site of the plasmid pcDNA3.1 + /Hygro (Invitrogen) containing both a CMV promoter and a polyadenylation signal derived from the bovine growth hormone gene (BGH pA). P1 and P2 indicate the relative positions of primer sequences used to generate a TF cDNA cassette encoding a truncated form of the protein lacking the cytoplasmic tail (TF^ΔTail). (B) The mutagenic P2 primer changed the CGC codon at position 934-936 in the Hartzell et al⁴⁴  numbering system to a termination codon (TAG) and introduced a downstream XhoI site to assist in molecular cloning (nucleotide substitutions denoted by *). The PCR product generated by these primers was digested with PpuMI and XhoI and ligated into a PpuMI/XhoI cut pcDNA3.1 + /Hygro/TF vector. (C) Diagnostic fluorescence-activated cell sorting analysis of murine TF expression on the surface of tumor cell lines using the ICON recombinant fusion protein containing murine fVII linked to the Fc region of IgG and a phycoerythin-conjugated Fc antibody. Note that a comparable pattern of TF expression was observed in TF^WT (green) and TF^ΔTail (red) cell populations based on fluorescence distribution and mean fluorescence intensity (59 and 61 arbitrary fluorescence units, respectively). Control analyses shown for comparison are TF^O cells processed in parallel for fluorescence-activated cell sorting analysis using ICON protein and phycoerythin-conjugated Fc antibody (gray) and TF^WT cells processed using the phycoerythin-conjugated Fc antibody, but in the absence of ICON protein (blue).