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Fig. 1. Targeted disruption of the Stra8 gene. (A) Structure of the targeting vector and partial restriction map of the WT Stra8 locus before [WT (+) allele] and after (L3 allele) homologous recombination, as well as after Cre-mediated excision (L– allele). Black boxes (1-5) indicate exons. The locations of restriction sites (E, EcoRI; N, NcoI; 47, Eco47III, X, XbaI) are indicated. Arrowhead flags represent loxP sites. neo, neomycin-resistance cassette; pBS-SK, pBluescript II SK+. Arrows indicate the location of primers #1, #2 and #3, which were used for genotyping. (B) Long-range PCR analysis of tail genomic DNA from mice with the indicated Stra8 genotype using primers #1 and #2 (upper panel), and #1 and #3 (lower panel). The identities of the different alleles are indicated on the right. Note that a large fragment corresponding to the (+) allele is amplified from DNA of WT mice using primers #1 and #3 (lower panel). The asterisk points to an unspecific product. (C) Schematic representation of the Stra8 transcripts that are synthesised from the WT or the mutant loci (left). Open and black segments in boxes 1-5 indicate non-coding and translated regions of the exons, respectively. The location of the ATG codon is specified for each Stra8 isoform. The size of the fragments amplified from the corresponding cDNA using primers #4 and #5 is also indicated. (Right) Results of RT-PCR assays obtained from P15 WT (+/+) and homozygous mutant (L–/L–) testis RNA using primers #4 and #5 (right panel). The size of the fragments is indicated on the right. Negative and positive controls were obtained using water (H2O) and a STRA8-expressing plasmid (pStra8), respectively. As expected, a shorter fragment corresponding to a truncated mRNA is amplified from the Stra8-null-testis RNA samples. The asterisk points to primer dimers.
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