FIG. 1.

 Generation and expression of the MTG8^Ex2/lacZ allele. (A) Diagram of the wt locus, the targeting vector and the mutant locus. Thick black line and boxes, murine MTG8; white box, lacZ; dark gray box, neo^r gene; light gray box, tk gene; thick striped lines, promoters (arrows indicate transcriptional starts) and 3′ UTR [lollipop symbols indicate poly(A) signals]; thin line, pUC18. The 5′ and 3′ bars indicate the positions of the probes flanking the targeted region and used in the Southern analysis. Relevant restriction sites are represented by capital letters (B, BamHI; H, HindIII; S, SstI, Sl, SalI). (B) Southern blotting analysis of wt (+/+) and MTG8^Ex2/lacZ-targeted (+/−) CCB ES cells. Restriction enzymes are as in panel A. The probes are described in Materials and Methods. The numbers on the left indicate the molecular size markers in kilobase pairs. (C) S1 protection analysis of brain RNA from wt (+/+) and MTG8 exon 2-null (−/−) pups with a probe spanning MTG8 exons 2 to 4 (Table 1). tRNA, negative control for probe hybridization. The top band corresponds to residual undigested full-length probe. The double filled arrows point to the band resulting from full protection of MTG8 sequences; the single filled arrow points to the band resulting from protection of exons 3 and 4 only. For reference purposes, protection by an actin probe added to the same hybridization mixture is shown at the bottom (single open arrow). The numbers on the left indicate the molecular size markers in nucleotides. (D) Western blotting analysis of brain from wt (+/+) and MTG8 exon 2-null (−/−) mice with an antiserum against the C-terminal domain of MTG8. No bands were visible with normal rabbit serum (data not shown). Asterisks mark species that are absent in the mutant. The numbers on the right indicate the molecular size markers in kilodaltons.