Fig. 2

Southern and immunoblot blot analysis of ES clones and animals

(A) Southern analysis of DNA from targeted ES clones, after XbaI digestion, and using a flanking probe (probe 1 from Fig. 1B). Lane 1, control J1 ES cell DNA; lanes 2–10, PCR-positive clones; +, wild-type allele (12 kb); KO, ld^GKO allele (2 kb). (B) Southern analysis of the same ES clones, after HindIII digestion, using an internal probe (probe 2 from Fig. 1B). +, wild-type allele (6 kb); KO, ld^GKO allele (8 kb). (C) Southern analysis of mice containing the ld^GKO allele. Tail DNA samples from the F2 offspring of a heterozygous cross (lanes 1–11) were analyzed by Southern blot analysis after EcoRI digestion, using the internal probe 2. +, wild-type allele (6 kb); KO, ld^GKO allele (8 kb). Pups 4, 5, 8, 9, and 11 were +/+, pups 1–3, 6, and 7 were ld^GKO/+, and pup 10 was ld^GKO/ld^GKO. (D) Immunoblot analysis of lysates from bodies, dissected away from head and limb buds, of E10.5 embryos of different genotypes. The genotypes analyzed were: ld^GKO/ld^GKO homozygotes (lanes 1,2); ld^GKO/ld^TgBri compound heterozygotes (lanes 3–6); and ld^GKO/+ heterozygote (lane 7). The relevant protein bands are indicated by arrows: IV refers to the 165-kDa isoform IV protein; B refers to the 155-kDa ld^TgBri truncated protein. A background band of about 120 kDa was shown to demonstrate relative protein loading in each lane.