Methylation-specific PCR analysis in Col8A1 promoter in Creole cattle carrier of rob(1; 29)

ABSTRACT

Robertsonian translocation (rob(1;29)) is the most frequent structural chromosomal abnormality in cattle. Heterozygous carriers have a normal phenotype but show a 3-5 percent decrease in fertility. Chromatin decondensation was evaluated similar to the inactive X chromosome when submitted to demethylating agent. Based on this result, and the concept that imprinted genes are essential in embryonic development, we decided to query genes located on BTA1 and BTA29 that could undergo genome imprinting. The collagen typeVIII- alpha 1 (Col8A1) acted on extracellular matrix structural proteins. DNA bisulfite conversion and sequentiation methods were used to compare its differential methylation patterns. It was performed on eight Creole cattle DNA blood samples from normal and rob(1;29) carriers. An in silico screening for CpG islands in its promoter uncovered a single region of 454 bp prone to methylation. BiQ-Analizer software was used to show the selective conversion of unmethylated cytosines to uracils obtaining the following results: unmethylated CpGs 0.000 (0 cases), methylated CpGs 0.802 (77 cases) and CpGs not present 0.198 (19 cases). No differences between samples were observed in this highly methylated region. This technique was successfully applied so it is a straightforward methodology that can be utilized to evaluate different tissue associated to specific gene expression.