Fig. 1. Mapping the gene-trap insertion in the Man1 locus. (A) Upper panel: the Man1 gene, showing exon organization (black boxes). Lower panel: the gene-trap vector, containing the engrailed2 intron (en2) and splice acceptor (SA), a ß-galactosidase-neomycin fusion cDNA (ß-geo), an SV40 polyadenylation site (SV40pA) integrated between exons 4 and 5. `Probe' indicates the location of the genomic probe used in Southern blotting. (B) Southern blot of genomic DNA from gene-trapped mice. Approximate size of detected DNA is indicated. (C) Northern blot, demonstrating 4.7 kb endogenous and 7.7 kb Man1/ß-geo transcripts in wild-type (+/+) or heterozygous (+/GT) MEFs. (D) The MAN1 911 amino acid polypeptide contains the LEM domain at the N-terminus, the RRM domain at the C-terminus and two transmembrane domains. The gene-trap insertion product retains the 567 N-terminal amino acids (arrow) linked to ß-galactosidase. (E) Western analysis on MEF whole cell extracts using an antibody raised to a C-terminus peptide shows that the C-terminus of MAN1 is absent in homozygotes (GT/GT). +/+, wild-type; +/GT, heterozygote; E, EcoRV.