Transmission and expression of mutant G-CSF-R. (A) Southern blot of EcoRV digests of tail DNA from awt/wt mouse (lane 1), a wt/▵715 mouse (lane 2), and a ▵715/▵715 mouse (lane 3) hybridized with probe b (Fig 1). (B) Nucleotide sequence analysis of PCR-amplified genomic DNA from a wt/wt and a▵715/▵715 mouse cloned in pBs. The sequence of the▵715-derived clone shows the C-to-T substitution changing CAG (Gln715) into TAG (stop715) and the silent base pair substitutions that generated the EcoRV site (GATATC). (C) Flow cytometry analysis of biotinylated G-CSF binding to BM cells from wt/wt,wt/▵715, and ▵715/▵715 mice. Cells were incubated in the absence (solid line) or presence (broken line) of a 100-fold molar excess of nonlabeled G-CSF followed by incubation with PE-conjugated streptavidin. (D) Western blot analysis of 1 × 106 BM cells from wt/wt and▵715/▵715 mice using a rabbit antiserum raised against the 20 C-terminal amino acids of the murine G-CSF-R. The three bands indicated with arrows represent the different glycosylation forms of murine G-CSF-R5; their absence in the▵715/▵715 lanes indicates that the C-terminus is truncated in the mutant mice. Reprobing with anti-STAT3 confirms equivalent loading in both lanes.
