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Fig. 6. Construction and analysis of a TAG-1 mutant allele that encodes N-terminally truncated TAG-1 protein. (A) Recombination strategy showing part of TAG-1 gene locus, including exon 2 (ATG and leader) and exons 3-13 (Ig domains 1-5), targeting construct and targeted TAG-1^a locus. Red lines indicate diagnostic BamHI fragments; black box, probe PP; arrowheads, polymerase chain reaction (PCR) primers. (B) Southern analysis of targeted ES cells with BamHI digest and probe PP. (C) PCR detection of wild-type (+/+) allele (orange primers in A) 450 bp product; TAG-1^a allele (green primers in A) 260 bp product. +/a, heterozygote; a/a, homozygote. (D) A major 135 kDa product detected by anti-TAG-1 in western analysis of +/+ and +/a mice, but not in a/a. Novel bands at 100 kDa and 110 kDa present only in +/a and a/a (see J). (E) Aberrant splicing of mRNA from TAG-1^a allele and primers (arrows) used for reverse-transcription (RT) PCR in F and G. (F) RT-PCR detects aberrant exon 2-7 splice using a and b primers (see E); 391 bp in a/a animals instead of normal 1017 bp product (+/+). (G) RT-PCR using a and c primers detects exon 2-9 splice (230 bp) in a/a instead of 1133 bp product (+/+). Faint band at 507 bp=exon 2-7 splice. (H) Cloning and sequencing of exon 2 to 9 product (a and d primers in E) confirms splice is in-frame. (I) Surface immunolabelling of wild-type and TAG-1^a/a sensory neurons with anti-TAG-1 (see Materials and methods). (J) Predicted proteins. Full-length TAG-1 mRNA encodes a 1041 aa protein, calculated MW 113 kDa (compare with observed MW of 135 kDa) (D). Exon 2-7 splice: 831 aa; calculated MW 89.9kDa; observed 110 kDa. Exon 2-9 splice: 740 aa; calculated MW 79.7 kDa; observed 100 kDa.

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