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For reproducible analyses of gene function in Xenopus, the use of transgenic strains is a promising approach but has limitations when investigating factors interfering with development. Therefore, inducible systems are attractive alternatives, and a binary system based on recombinases is a most versatile approach. We have shown previously that Cre and FLP recombinases are active in Xenopus laevis and can induce a silent reporter gene in a corresponding reporter strain. Here, we describe the establishment of the transgenic Xenopus laevis strain A7 expressing Cre recombinase under the control of the muscle-specific cardiac actin promoter. Upon crossing to several distinct reporter strains, A7 is able to induce EYFP, DsRed2, or LacZ reporter genes in a muscle-specific manner. This first Cre-expressing strain allows conditional activation of any gene of interest in muscle cells and, thus, opens up the use of recombinases as a new experimental strategy in Xenopus.
Figure 2. Recovery of the A7 CAR:Cre strain. Frozen sperm from the A7 male was microinjected into eggs derived from the C5 reporter strain containing the LCMV:ECFP(loxP)(FRT)EYFP transgene. A: Stage 42 tadpole showing blue fluorescence of the ECFP marker gene. B: The same tadpole in the yellow filter set shows yellow fluorescence in tailmyotomes. C: A Cre-positive tadpole with the activated EYFP reporter gene in myofibrils of the jaws and the abdominal wall. Stage 52, ventral view. D: Tail-tip polymerase chain reaction analysis for the presence of Cre in tadpoles with or without activation of the reporter gene in muscle cells. Tadpoles from the same crossing without the reporter gene were also analyzed. Scale bars = 1 mm.Download figure to PowerPoint
Figure 3. Crossing a recovered A7 F1 male with the C5 reporter strain. Larvae obtained from this crossing were selected for blue fluorescence, indicating the presence of the reporter gene LCMV:ECFP(loxP)(FRT)EYFP. A: Lateral view and ventral view of the head of a Cre-positive tadpole in the blue filter set. B: The same tadpole in the yellow filter set shows yellow fluorescence in tailmyotomes and myofibrils of the jaws. C: Lateral view and ventral view of the head of a Cre-negative tadpole in the blue filter set. D: The same tadpole in the yellow filter set displays fluorescence that represents the bleed-through of the CMV driven ECFP activity. E: A Cre-positive tadpole at a later stage, ventral view. Left panel: blue filter set. Right panel: yellow filter set. No yellow fluorescence was observed in the heart. F: A Cre-positive tadpole, close up from the head, dorsal view. Left panel: blue filter set. Right panel: yellow filter set. Myofibrils near the eye show activation of the EYFP reporter gene. Scale bars = 1 mm.Download figure to PowerPoint
Figure 4. Crossing a recovered A7 F1 female with the G/R reporter strain. Larvae obtained from this crossing were selected for green fluorescence, indicating the presence of the reporter gene CAG:EGFP(loxP)DsRed2. A: Lateral view of a Cre-positive tadpole in the green filter set (upper panel). The same tadpole in the red filter set shows red fluorescence in tailmyotomes (lower panel). Unspecific activation of red fluorescence was also observed (arrows). B: A Cre-negative tadpole showing mosaic expression of the CAG promoter driven EGFP gene (upper panel) and no red fluorescence in the red filter set (lower panel). C,D: Examples for induction of red fluorescence as well as induction of green fluorescence in muscle cells at a later developmental stage. C: Cre-positive larvae showing activation of green fluorescence in tailmyotomes (upper panel, left) as well as activation of the red fluorescent reporter gene (lower panel, left). The right-hand panels show close-ups from myofibrils of the jaws in the respective filter sets. D: A Cre-negative sibling showing background green fluorescence in tailmyotomes (upper panel) and lack of red fluorescence (lower panel). Scale bars = 1 mm.Download figure to PowerPoint
Figure 5. Crossing a recovered A7 F1 male with the GPL8 reporter strain. Larvae obtained from this crossing were selected for green fluorescence, indicating the presence of the reporter gene CMV:GFP(loxP)LacZ. A–C: A Cre-positive tadpole showing LacZ staining in muscle cells. A: Before LacZ staining, green filter set. B: After LacZ staining, blue myotomes are detected. C: Diffuse staining was observed in the heart. D–F: A Cre-negative tadpole before LacZ staining (D), green filter set. E: After LacZ staining, lateral view. F: After LacZ staining, ventral view. G–I: Close-up of a Cre-positive larvae in the tail showing incomplete staining of the myotomes (G), in the head showing incomplete LacZ staining of the myofibrils of the jaw (H), and in the head region near the eye (I). Examples for unspecific LacZ staining are indicated by arrows. K: Polymerase chain reaction analysis for the presence of the Cre transgene in tadpoles with or without activation of the lacZ reporter gene in muscle cells. wt, wild-type tadpole as negative control. Scale bars = 1 mm in A–H, 100 μm in I.Download figure to PowerPoint
GpL8: Contains the Cre reporter CMV:ECFP(loxP)LacZ.
The larvae show green fluorescence that upon Cre recombinase action turns into beta-galactosidase activity that can be visualized by LacZ staining.
