Improved cre reporter transgenic Xenopus

Abstract

We have produced and characterized improved transgenic reporter lines for detection of Cre recombinase activity during Xenopus development. Improvements include choice of fluorophores, which make these Cre reporter lines generally suitable for lineage tracing studies. We also include data for several new parameters affecting survival and transgenesis efficiency using the recently developed meganuclease method of frog transgenesis. These transgenic frogs express cyan fluorescent protein (CFP) under control of the ubiquitous promoter CMV, where CFP is replaced by DsRed2 (a red fluorescent protein) in the presence of Cre. Three independent, high expression, Cre-sensitive lines have been identified that maintain robust fluorophore expression across generations and lack DsRed2 expression in the absence of Cre. A novel use of these lines is to indelibly mark embryonic blastomeres by Cre mRNA injection for permanent fate mapping. Similarly, transgenically expressed Cre under control of tissue-specific promoters will allow detailed analysis of cell lineage relationships throughout embryogenesis, metamorphosis, and adulthood.

Figure 1

Constructs used for transgenesis. Gray arrows represent 18 base pair SceI recognition sequence with arrowhead at larger downstream cleavage product. In pDR>CG<-SceI, SceI will remain bound to the portion of the plasmid containing CRY:DsRed1 (the “inside” portion) but not CMV:EGFP (the “outside” portion) because downstream ends of SceI sites are oriented towards the CRY:DsRed1 cassette. CFP-cyan fluorescent protein, CMV-cytomegalovirus ubiquitous promoter, CRE-Cre recombinase, CRY-gamma crystallin lens-specific promoter, DsRed- Discosoma sp. red fluorescent protein, EGFP- enhanced green fluorescent protein, GFP3- green fluorescent protein 3, HSP- heat shock-inducible promoter, loxP- Cre recombinase recognition site, YFP- yellow fluorescent protein.

Figure 2

Exemplar tadpole fluorescence patterns. Top: Tadpole injected with pDR>CG

Figure 3

Cre activity in pCLFR-SceI in F0 transgenic tadpole. Tadpole subjected to meganuclease injection followed by Cre mRNA injection at the one cell stage is half transgenic for pCLFR-SceI as visualized in the CFP filter set. Red fluorescence in the RFP filter set indicates Cre activity removed the CFP to allow DsRed2 expression. The CFP expression is not completely extinguished, especially in the gill cartilages (arrow) for unknown reasons. No embryos lacking Cre mRNA injection showed red fluorescence, indicating that Cre expression is required to allow DsRed2 expression. The strong blue signal in the head region is likely from episomal expression. Autofluoresence can be seen in the intestinal coils and gall bladder.

Figure 4

F1 offspring of pCLFR-SceI founder. Brightfield (top), blue fluorescence (middle), and red fluorescence (bottom) showing transgenic and non-transgenic siblings from Male #4. No red fluorescence was seen in F1 offspring in the absence of Cre activity.

Figure 5

Lineage tracing via Cre mRNA injection into pCLFR-SceI transgenic embryos. Cre mRNA was injected into one cell of two-cell stage embryos (A) or the A1 blastomeres of the 32-cell stage embryo (B). (A) Brightfield, CFP, and RFP images show half of the body is exclusively blue, and the other half is mostly red with a few blue cells. The replacement of blue by red fluorescence is particularly evident in brain and tail. (B) Two examples of tadpoles showing results when both (left) or just one (right) A1 blastomere was injected with Cre mRNA. Intestine contains yolk autofluorescent in both filter sets. Insets show diagrams of injections.

Figure 6

Transgenic expression of Cre in pCLFR-SceI induces blue to red conversion. Meganuclease transgenesis procedure was performed with co-injection of pCSCRE2 lacking SceI sites to allow mosaic expression of Cre. Brightfield image shows mid-section of tapole with portions of abdomen and tail representing region of tadpole shown in all panels. Control tadpole lacks transgenic fluorescence and only autofluorescent yolk is seen. The pCLFR-SceI tadpole shows blue only, whereas mosaic transgenic expression of Cre resulted in several cells showing red fluorescence indicative of Cre-mediated switch from CFP to DsRed2 expression.