Production of an aberrant splice variant of CCL5 is not caused by genetic mutation in the mammary glands of mastitis-infected Holstein cows

Genetic mutations, including single nucleotide polymorphisms (SNPs), result in aberrant alternatively splicing of gene and involves in susceptibility of inflammatory diseases, including bovine mastitis. C-C motif chemokine ligand 5 (CCL5) is an immune-associated gene, but its alternative splicing (AS) mechanism of gene expression has not yet been understood. The present study identified the splice variant of CCL5 and the compared differential expression of various transcripts between healthy and mastitic mammary gland tissue from cows. A novel transcript lacking exon 2 with a deletion of 112 bp (referred to as CCL5-AS) was identified in the mammary gland. The expression of CCL5-AS was lower compared with CCL5-reference in the healthy and mastitic mammary tissues. A total of two novel SNPs (g.1647 C>T and g.1804 G>A) were identified in exon 2 of CCL5. Using the splicing mini-gene reporter assay in bovine mammary epithelial cells (MAC-T) and 293T cells, it was confirmed that the production of CCL5-AS was not caused by the two SNPs. The present findings suggested that alternative splicing is one of the mechanisms of CCL5 expression regulation and is involved in mastitis infection, but that genetic mutation was not responsible for the generation of the abnormal transcript of CCL5 in cows.