Identification of a Splice Variant of gp100 Expressed in Canine Melanoma Tumours

Abstract

Introduction: The melanoma‐associated antigen (MAA) gp100 is a glycoprotein expressed by melanocytes and is considered to be an important target for anti‐tumour immunity in human melanoma. The aim of the current study was to characterise the canine gp100 gene with a view to its inclusion in a DNA vaccine for treatment of canine oral malignant melanoma.Methods: RNA was extracted from eight canine oral melanomas and seven control samples (pigmented oral mucocutaneous tissue) and cDNA synthesised. PCR was performed using human gp100 primers designed to generate a 421base pair (bp) fragment. In addition to the expected pcr product, a smaller amplicon (∼260 bp) was present in all tumours and control tissues. Both PCR amplicons were cloned and sequenced. The gp100 genomic DNA sequence was identified from the dog genome resource (NCBI).Results: The 421 bp fragment of canine gp100 shared 90.8% identity with human gp100. This partial sequence was located on dog chromosome 10 (CFA10_contig 2982). Further analysis, using the human gp100 gene as a reference, allowed the complete canine gp100 coding sequence to be identified from the dog genome. Sequence analysis of the small PCR product showed it to be a splice variant of gp100 with exon 5 deleted.Conclusion: The canine gp100 sequence has been identified and this will allow DNA vaccine constructs containing this MAA to be developed. The splice variant of gp100 with exon 5 deleted was not tumour‐specific and was expressed in both melanomas and normal pigmented tissue.