Species Authentication of an Endangered Sheep Fibroblast Biobank Using a Multi-Gene PCR Approach

A study was carried out to characterize and establish ear pinna-derived fibroblast cell lines from Katchaikatti Black sheep by species identification using a candidate gene approach. Classical polymerase chain reaction (PCR) assays targeting the D-loop of mitochondrial DNA, prolactin and ?-casein genes of nuclear DNA were performed using genomic DNA isolated from dermal fibroblast cells of Katchaikkatti Black sheep at passage level 3. PCR-amplified DNA fragments were cloned into E. coli-based vector and the confirmed recombinant plasmids were subjected to gene sequencing. The obtained nucleotide sequences of three genes of sheep were analysed using the Clustal bioinformatics tools. It was found that the obtained nucleotide sequences of the three genes had 98 to 99% identities with the corresponding gene sequences of Ovis aries. In the phylogenetic tree analysis based on the D-loop, Katchaikatti Black sheep formed a unique branch. It was concluded that the established cell lines are species-specific, devoid of cross-contamination, and could be used for further downstream applications.