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

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.

SUBMITTER: Biegler MT

PROVIDER: S-EPMC8921232 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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

Biegler Matthew T MT Fedrigo Olivier O Collier Paul P Mountcastle Jacquelyn J Haase Bettina B Tilgner Hagen U HU Jarvis Erich D ED

Scientific reports 20220314 1

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, all ...[more]

PMID: 35288582

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Project description:Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.

Dataset Information

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

Publications

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

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