Temporally-Controlled Site-Specific Recombination in Zebrafish

Stefan Hans,Michael Brand,Jan Kaslin,Dorian Freudenreich,Darren P Martin

doi:10.1371/journal.pone.0004640

Abstract

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

Highlights

Zebrafish (Danio rerio) is a widely used model organism for vertebrate development and disease [1] due to its short generation time, large number of offspring and optical clarity which allow large-scale forward mutagenesis screens with relative ease [2,3]
In the absence of Cre activity the reporter line expresses DsRed2 under the control of the Xenopus Elongation Factor 1 alpha (EF1a) promoter, but changes to EGFP after a successful recombination event (Fig. 1a,b)
Following a brief heat induction at mid-gastrulation stages that results in only weak EGFP fluorescence derived from the Tg(hsp70:EGFP-Cre) allele, the DsRed2 signal is reduced and strong ubiquitous EGFP expression can be observed in double transgenic embryos (Fig. 1e,f)

Summary

Introduction

Zebrafish (Danio rerio) is a widely used model organism for vertebrate development and disease [1] due to its short generation time, large number of offspring and optical clarity which allow large-scale forward mutagenesis screens with relative ease [2,3]. A complementary reverse genetic application is the temporal and spatial controlled over-expression of genes using the Gal4-UAS [8] and the mifepristone-inducible LexPR system [9]. Once the transcriptional activator has vanished, expression of the gene of interest is lost, impeding genetic fate mapping in zebrafish. Based on the observation that the localization of proteins can be controlled by a ligand when fused to a ligand-binding domain of a steroid hormone receptor, chimeric Cre recombinases were developed [12]. This approach offers temporal control of Cremediated recombination and allows targeting late aspects of a dynamic or broad Cre expression. Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) has the best properties for ligand sensitivity and inducible recombination efficiency [13]

Methods

Results

Conclusion