AbstractThe zebra finch is one of the most commonly studied songbirds in biology, particularly in genomics, neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. We generated a cell line, designated CFS414, from zebra finch embryonic fibroblasts using the SV40 large and small T antigens. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. Using this cell line, we disrupted endogenous gene sequences using S.aureus Cas9 and confirmed a stress-dependent localization response of a song system specialized gene, SAP30L. The utility of CFS414 cells enhances the comprehensive molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.