Abstract
The 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.
Highlights
The zebra finch is one of the most commonly studied songbirds in biology, in genomics, neuroscience and vocal communication
We further demonstrated the cell line’s applicability to test guide RNAs for CRISPR-mediated genome editing and study genes related to vocal learning and other songbird traits
The presence of SV40Tt in CFS414 genomic DNA was confirmed with PCR (Fig. 1F)
Summary
The zebra finch is one of the most commonly studied songbirds in biology, in genomics, neuroscience and vocal communication This species lacks a robust cell line for molecular biology research and reagent optimization. Zebra finch cell lines have been established using an induced pluripotent stem cell (iPSC) construct, STEMCCA, containing four transcription factors[13], or from tumors (G266 and ZFTMA) from a male and female, r espectively[14] These lines are difficult to maintain as culture stocks, compared to stable cell lines from other species, and have not been widely utilized across the avian science community. Other avian cell lines include a duck embryonic fibroblast line, DEF-TA, that was immortalized with the Simian vacuolating virus 40 (SV40) large T a ntigen[24] Each of these cells are capable of robust, density-independent survival and proliferation, enabling clonal propagation and antibiotic selection strategies. As we learn more about both the convergent and unique biological properties of songbirds[16,26,27], the need for immortalized cell lines becomes increasingly clear
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