Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

Jung, Kyung Min; Kim, Young Min; Kim, Jin Lee; Han, Jae Yong

doi:10.1038/s41598-021-94229-x

Cited by 7 publications

(6 citation statements)

References 46 publications

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“…Also, the large transduction efficiency and titers of adenovirus are significant advantages when a limited volume of adenovirus, ∼2 μL, can be administered in the confined subgerminal cavity. Furthermore, because the transduction of turkey and zebra finch in vitro by adenovirus has been reported ( 9 , 16 ), the adenovirus-mediated method can be utilized to produce genome-edited birds in these avian species. Based on these advantages, adenovirus type 5 is a very efficacious vehicle for delivering the CRISPR-Cas9 system to avian blastodermal cells in vivo for inducing genome editing efficiently and conveniently in blastodermal PGCs to produce genome-edited birds of different species.…”

Section: Resultsmentioning

confidence: 99%

Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing

Lee

Kim

Karolak

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.

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Section: Resultsmentioning

confidence: 99%

Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing

Lee

Kim

Karolak

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…The use of genome editing tools such as CRISPR-Cas9 has only recently been reported in songbirds 40 , in part due to the lack of robust genetic substrates for testing and optimizing these tools. To assess editing efficiency in the zebra finch genome using CFS414 cells, we generated a two-plasmid system for CRISPR-mediated gene targeting.…”

Section: Resultsmentioning

confidence: 99%

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

Biegler

Fédrigo

Collier

et al. 2022

Sci Rep

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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.

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“…The electroporation-mediated transfection efficiency of primary ZEFs has been reported previously as 40%, with a cell viability of less than 50%, and the gene editing efficiency has been reported as up to 30% [ 31 ]. The immortalized cell line described here showed an improved lipofection-based transfection efficiency of approximately 50%, with high cell viability.…”

Section: Discussionmentioning

confidence: 99%

Generation and characterization of genome-modified chondrocyte-like cells from the zebra finch cell line immortalized by c-MYC expression

et al. 2022

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Background Due to their cost effectiveness, ease of use, and unlimited supply, immortalized cell lines are used in place of primary cells for a wide range of research purposes, including gene function studies, CRISPR-based gene editing, drug metabolism tests, and vaccine or therapeutic protein production. Although immortalized cell lines have been established for a range of animal species, there is still a need to develop such cell lines for wild species. The zebra finch, which is used widely as a model species to study the neurobiological basis of human speech disorders, has been employed in several functional studies involving gene knockdown or the introduction of exogenous transgenes in vivo; however, the lack of an immortalized zebra finch cell line has hampered precise genome editing studies. Results Here, we established an immortalized cell line by a single genetic event, expression of the c-MYC oncogene, in zebra finch embryonic fibroblasts and examined its potential suitability for gene targeting investigations. Retroviral vector-mediated transduction of c-MYC was used to immortalize zebra finch primary fibroblasts; the transformed cells proliferated stably over several passages, resulting in the expression of chondrocyte-specific genes. The transfection efficiency of the immortalized cells was much higher than that of the primary cells. Targeted knockout of the SOX9 gene, which plays a role in the differentiation of mesenchymal progenitor cells into chondrocytes, was conducted in vitro and both apoptosis and decreased expression levels of chondrogenic marker genes were observed in edited cells. Conclusions The c-MYC induced immortalized chondrocyte-like cell line described here broadens the available options for establishing zebra finch cell lines, paves the way for in-depth biological researches, and provides convenient approaches for biotechnology studies, particularly genomic modification research.

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Efficient gene transfer into zebra finch germline-competent stem cells using an adenoviral vector system

Cited by 7 publications

References 46 publications

Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing

Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing

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

Generation and characterization of genome-modified chondrocyte-like cells from the zebra finch cell line immortalized by c-MYC expression

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