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CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish

Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes...

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Bibliographic Details
Main Authors: Auer, Thomas (Author) , Duroure, Karine (Author) , Concordet, Jean-Paul (Author) , Del Bene, Filippo (Author)
Format: Article (Journal)
Language:English
Published: 13 November 2014
In: Nature protocols
Year: 2014, Volume: 9, Issue: 12, Pages: 2823-2840
ISSN:1750-2799
DOI:10.1038/nprot.2014.187
Online Access:Resolving-System, lizenzpflichtig, Volltext: https://doi.org/10.1038/nprot.2014.187
Verlag, lizenzpflichtig, Volltext: https://www.nature.com/articles/nprot.2014.187
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Author Notes:Thomas O. Auer, Karine Duroure, Jean-Paul Concordet, Filippo Del Bene
Description
Summary:Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.
Item Description:Gesehen am 14.10.2020
Physical Description:Online Resource
ISSN:1750-2799
DOI:10.1038/nprot.2014.187

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