EIIIA^–/– and EIIIA-flox mice are viable and maintain expression and extracellular matrix association of FN. (A) Top line: EIIIA targeting vector with loxP sites depicted by black triangles, selection cassettes, and the EIIIA exon. Second line: wild-type EIIIA locus with hybridization probes indicated by p1 and p2. Third line: targeted allele. Fourth line: EIIIA^–/– and EIIIA-flox alleles resulting from Cre-loxP–mediated recombination between sites 1 and 3 or sites 1 and, 2 respectively. Restriction enzyme sites BamHI (B), EcoRI (R), HindIII (H), and SmaI (S) are indicated, and restriction fragments are shown by dashed lines. (B) Southern blot analysis of wild-type (^+/+) and heterozygous (^+/–) EIIIA^–/– and EIIIA-flox embryonic stem (ES) cell DNA, hybridized with probe 2. (C) Southern blot analysis of mouse tail-tip DNA, from progeny of EIIIA^–/– or EIIIA-flox heterozygotes, using probe 2. Homozygotes are indicated by ^–/–. (D) RT-PCR analysis of alternative exons EIIIA, EIIIBm and V/CS-1 expression, using primers that flank each. (+) mRNA indicates inclusion and (–) mRNA indicates exclusion of the exon. (E) Immunofluorescence staining of EIIIA-FN or total FN on wild-type and EIIIA^–/– fibroblasts. Original magnification, × 200. (F) Western blot analysis of total FN in DOC-soluble and -insoluble matrix from wild-type and EIIIA^–/– fibroblasts at 4 and 24 hours after plating. Neo indicates neomycin-resistance gene (Neo); herpes simplex virus thymidine kinase gene (TK).