Werdien D et al. (2001), FLP and Cre recombinase function in Xenopus emb...

XB-ART-8984

Nucleic Acids Res 2001 Jun 01;2911:E53-3. doi: 10.1093/nar/29.11.e53.

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FLP and Cre recombinase function in Xenopus embryos.

Werdien D , Peiler G , Ryffel GU .

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The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.

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	Figure 1. Constitutive FLP activity in Xenopus embryos. (A) The FLP reporter construct CMV:GFP(FRT)lacZ is schematically drawn with the CMV promoter/enhancer driving the expression of the GFP and lacZ genes. The position of the SV40 early (EpA) and late (LpA) polyadenylation signals as well as the FRT target sites of the FLP are given. (B and C) Stage 36 Xenopus larvae were derived from eggs injected with CMV:GFP(FRT)lacZ DNA (B) or coinjected with CMV:GFP(FRT)lacZ DNA and FLPe mRNA (C). The living animals were photographed in normal light (top) or in fluorescence light (middle). X-gal staining was obtained after fixation of the larvae (bottom).
	Figure 2. 4-Hydroxytamoxifen-regulated FLP activity in Xenopus embryos. (A and B) Stage 30 Xenopus larvae were derived from fertilized eggs coinjected with CMV:GFP(FRT)lacZ DNA and FLPER(T) mRNA. Embryos were cultured either in the absence (A) or in the presence of 4-hydroxytamoxifen (B). The living embryos were photographed in normal light (top) or in fluorescence light (middle). X-gal staining was obtained after fixation of the larvae (bottom).
	Figure 3. FLP reporters encoding different fluorescent proteins. The FLP inducible reporters CMV:EYFP(FRT)ECFP (A), CMV:ECFP(FRT)EYFP (B), CMV:DsRed(FRT)GFP (C) and CMV:GFP(FRT)DsRed (D) are schematically drawn in each panel. Xenopus larvae were derived from eggs injected with reporter DNA (left panels) or coinjected with reporter DNA and FLPe mRNA (right panels). The living larvae were photographed in normal light (top picture in each panel) or in fluorescence light using defined filter sets (middle and bottom picture in each panel). The excitation/barrier filters were 480/510 nm for EYFP and GFP, 425/475 nm for ECFP and 546/590 nm for DsRed.
	Figure 4. Constitutive Cre activity in Xenopus embryos. (A) The Cre reporter construct CMV:GFP(loxP)lacZ is schematically drawn with the CMV promoter/enhancer driving the expression of the GFP and lacZ genes. The position of the SV40 EpA and LpA signals as well as the target sites of the Cre recombinase (loxP) are given. (B and C) Stage 38 Xenopus larvae were derived from eggs injected with CMV:GFP(loxP)lacZ DNA (B) or coinjected with CMV:GFP(loxP)lacZ DNA and Cre mRNA (C). The living animals were photographed in normal light (top) or in fluorescence light (middle). X-gal staining was obtained after fixation of the larvae (bottom).

References [+] :

Amaya, A method for generating transgenic frog embryos. 1999, Pubmed, Xenbase