January 1, 2006;
I-SceI meganuclease-mediated transgenesis in Xenopus.
Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.
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Figure 1. Generation of transgenic frog embryos with various promoters using I-SceI meganuclease. A–C: Transgenic embryos were generated with the ubiquitous promoters pCMV-GFP-SceI (1 and 2) and pCSKA-GFP-SceI (3 and 4; A); the lens-specific promoter pCry1GFP-SceI (B); and the pancreas-specific promoter pElastase-GFP-SceI (C).
Figure 2. a–d: F1 offspring obtained from F0 founder frog line 2 darkfield (a) and brightfield (c), and line 3 darkfield (b) and brightfield (d), showing strong and specific green fluorescent protein expression in the pancreas.
Fig 3. Southern blot analysis of transgenic lines. A: Schematic diagram of pElastase-GFP-SceI containing a pancreas-specific promoter and a green fluorescent protein (GFP) coding region. B: Southern blot analysis was performed with genomic DNA isolated from F1 offspring of four F0 founder frogs (lines 2-5) injected with pElastase-GFP-SceI and scored positive for GFP in the pancreas. The a and b indicate two different individuals from the same line. Genomic DNA was digested with BamHI and hybridized with insert probes resulting from NotI digestion. The copy number of integrated DNA was estimated using a dilution series (as indicated) of plasmid DNA digested with BamHI. 2X-64X, plasmid standards. Quantities indicated on top of each lane correspond to 2 to 64 copies of the plasmid integrated into the genome.