PUBLICATION

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

Authors
Auer, T.O., Duroure, K., Concordet, J.P., Del Bene, F.
ID
ZDB-PUB-141114-1
Date
2014
Source
Nature Protocols   9: 2823-2840 (Journal)
Registered Authors
Auer, Thomas, Del Bene, Filippo, Duroure, Karine
Keywords
none
MeSH Terms
  • Animals
  • Animals, Genetically Modified*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Embryo, Nonmammalian
  • Gene Knock-In Techniques
  • Genes, Reporter
  • Genetic Engineering/methods*
  • Green Fluorescent Proteins/genetics*
  • Transgenes
  • Zebrafish/embryology
  • Zebrafish/genetics*
PubMed
25393779 Full text @ Nat. Protoc.
Abstract
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.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping