FIG. 1.

Generation and screening of calnexin gene-deficient mice. (a) Map of calnexin germ line configuration, the targeting vector, the targeted allele with selectable markers, and the targeted allele after removal of the selectable markers. Restriction enzyme sites are indicated by single letters as follows: A, ApaI; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SmaI. (b) Southern blot screening of representative mouse tail biopsy specimens showed correct recombination at the 5′ calnexin locus. wt, wild type; he, heterozygous; ko, knockout. (c) Western blot analysis with antibodies (α) against the N terminus and C terminus of calnexin confirmed the absence of calnexin protein in calnexin gene-deficient mice. Additional, low-molecular-weight bands were observed on the Western blots. These are most probably nonspecific, since they were variably observed in different experiments. (d) Calrecticulin levels are unaltered in the absence of calnexin. (e) A second, independent calnexin gene-deficient mouse strain, with selectable markers deleted, expressed a truncated protein 15 kDa smaller than full-length calnexin because exons 4 to 6 were missing. The deletion included regions required for the glucose-binding pocket; the ER-targeting region was still expressed.