FIG. 3.

ACLP genomic organization and targeted disruption in mice. (A) The mouse ACLP gene contains 21 exons. Exons 11 through 14 encode the discoidin domain, while exons 15 through part of 21 encode the carboxypeptidase-like domain. A targeting construct was designed to replace exons 7 through 16 with a neomycin selection cassette (NEO). E, EcoRI; H, HindIII; B, BamHI; S, SalI; S, SacI. (B) Genotypes were determined by Southern blot analysis of EcoRI-digested genomic DNA with probes flanking the targeting construct (5′ or 3′ probes). The wild-type allele is ∼19 kb, and the targeted allele, detected with the external 5′ or 3′ probes, is smaller due to the internal EcoRI site in the NEO cassette. (C) Northern analysis of RNA from 14.5-dpc mouse embryonic fibroblasts. 18S rRNA oligonucleotide hybridization was used as an RNA loading control. (D Western analysis with an ACLP antiserum directed against the carboxyl terminus confirmed that the absence of ACLP mRNA correlated with the absence of ACLP protein in the knockout embryos.