Sang, Y., Ortega, M.T., Blecha, F., Prakash, O. and Melgarejo, T.
Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM®-T Vector, which was then linearized and treated with exonuclease III to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)