Induction of an immortalized songbird cell line allows for gene characterization and knockout by CRISPR-Cas9

The zebra finch is a powerful model for several biological fields, particularly neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. Here we describe a cell line, CFS414, generated 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 demonstrated a stress-dependent localization response of the zebra finch song nuclei specialized gene, SAP30L, and disrupted endogenous gene sequences using S.aureus Cas9. The utility of this cell line enhances the molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.