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. 2009 Nov 6;5(11):e1000712. doi: 10.1371/journal.pgen.1000712

Figure 1. Generation of OAZ-t knockout mice.

Figure 1

(A) Schematic representation of the methods used for gene targeting of the OAZ-t genome. The gene targeting construct contains Neo (open box) between the 4-kb 5′-arm and 9-kb 3′-arm (thick lines). As a result, exons 1–5 were replaced with Neo. Exon 1 includes the first methionine. Arrows indicate the transcriptional direction of OAZ-t. S indicates SacI restriction sites. (B) The targeted allele was identified by the Southern blotting of genomic DNA digested with SacI using a probe created from the 3′ fragment. (C) The mice were genotyped by PCR using two sets of primers: one set for the amplification of the Neo gene (Neo) and one set for the amplification of the OAZ-t gene. +/+: wild-type; +/−: heterozygous mutant; −/−: homozygous mutant. (D) Analysis of gene expression by northern blotting. No OAZ-t transcripts were detected in the testes of OAZ-t homozygous mutant mice. The same membrane was rehybridized with AZ-1 or Gapdh cDNA as a control. (E) Western blotting of testicular lysates from adult mice using anti-OAZ-t polyclonal antibodies. OAZ-t was not detected in the lysates from the homozygous mutant mice (22 kDa). The 19-kDa band may be a degradation product of OAZ-t. The asterisk indicates a non-specific band. β-Actin (ACTB) was used as a control.