A Survey of the Species Origins of Common Fish Cell Lines—With a Particular Focus on the Grass Carp Ovarian Cell Line ( <scp>GCO</scp> )

Cell line misidentification or contamination undermines the reliability of research outcomes. In this study, we verified the species origin of commonly used fish cell lines, with a particular focus on the grass carp ovarian cell line (GCO). RNA-seq data from 146 samples of 11 widely used fish cell lines were collected from public databases (NCBI-SRA) and were mapped to the reference genomes of eight species. GCO RNA-seq samples from three Chinese laboratories showed a mapping rate of 21.64% ± 3.61% to the grass carp genome, but 89.81% ± 4.83% to the fathead minnow genome. Further transcriptomic similarity analysis revealed that these GCO RNA-seq samples clustered more closely with cell lines originating from fathead minnow. Targeted analysis of the COI gene amplification region confirmed that nearly all reads from GCO RNA-seq samples mapped exclusively to the fathead minnow COI amplicon. Collectively, these results indicate that GCO may have undergone cross-contamination or species misidentification. This study raises doubts about the true species origin of the GCO cell line and proposes that COI DNA barcoding can be used to discriminate the species origin of fish cell lines.