Figure 1. Generation of a floxed Rab7 allele. (A) Targeting strategy for rab7 deletion in mice. Arabic numbers indicate primer pairs used to specifically amplify the alleles. Roman numerals correspond to exons. WT, wild type; H, HindIII restriction sites; solid triangles, LoxP sites; NEO, neomycin resistance gene. (B) PCR amplification of genomic DNA from MEFs of the indicated genotype. The positive control in the upper panel is tail DNA from a Rab7^+/flox mouse and in the lower panel genomic DNA isolated from T cells of a rab7 TKO mouse. (C) Western blot of lysates prepared from Rab7^+/+ and rab7^−/− MEFs. (D) Rab7^+/+ and rab7^−/− MEFs were fixed and permeabilized, stained for RAB7, and analyzed by flow cytometry. The secondary antibody alone (2° Ab) reflects nonspecific staining, results from all cells are shown. (E) Rab7^+/+ or rab7^−/− MEFs were evaluated by immunofluorescence microscopy.