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Method of germline transgenesis using transposons in Xenopus

Using the Sleeping beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos.

We have tested different transposons containing the GFP fluorescent marker, under the control of either ubiquitous promoters (pCMV), or of the muscle-specific promoter (pCar). These transposons were usually injected in the embryos as circular plasmids. We have compared the fluorescence at different developmental stages of animals injected with plasmids with or without transposase mRNA. The fluorescence is essentially mosaic and always visible in caudal muscle fibers, whatever the promoter was used. As fluorescence is also present in the control without transposase, it is not possible in the early stages to conclude for integration of the transposon in genomic DNA.

However, very interesting results were obtained with a particular construct in which the GFP gene is interrupted by an intron and under the control of the chicken ßactin promoter. In addition to the mosaic and clonal patterns of expression, we got a reasonable number of "half-transgenic" animals, which are fully fluorescent on either the left or the right side of the organism. This indicates integration of the GFP gene in one of the blastomers at the 2-cell stage. Altogether, 38% of the tadpoles where highly fluorescent mosaics or half-transgenics, compared to 12% in the absence of transposase, suggesting integration by transposition.

We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. Although the transposase is necessary for efficient generation of transgenic animals, the analysis of excision reveals non-canonical molecular footprints. The canonical footprint is a trinucleotide C(A/T)G between two TA dinucleotide. The sequence of several plasmids indicates that excision of the transposon has occurred, but the size and the position of the borders of the excised fragment are variable. This phenomenon has also been observed in the mouse. The possible explanations for these non-canonical footprints could involve either a precise excision of the transposon followed by different plasmid repairs like those found in mice, or a non-properly excision of the transposon from the donor plasmid. These results are in agreement with previously-reported data showing that SB transposon leaves characteristically different footprints at excision sites in different cell types.

Nevertheless, the important conclusion of this analysis is that the transposase is active in the embryo, since it is able to catalyse the first step of the transposition process.
We investigated the molecular nature of integrations by Southern blot analysis on two F0 animals, from lines exhibiting two different levels of fluorescence, and on their offsprings. These results proved that the transgene integration did not occur though a canonical cut and paste transposition mechanism for both lines. Our results lead us to suggest that X. laevis embryos do not possess all the co-factors necessary to mediate precise integration by transposition. This could be the consequence of a non-canonical cell cycle during Xenopus early segmentation, during which DNA repair does not occur. Surprisingly, non expressing F1 siblings were also positive for hybridization with GFP and plasmid probes. These data suggest that several integrations of the transgene occurred in F0 germline, but that some of them are silenced by position effects.

From the present study, we conclude that SB transposase can be used with some advantages to obtain numerous transgenics, and specifically hemi-transgenic animals, but we cannot assess if the integration of the transgene does result from imperfect transposition events or from other DNA recombination mechanisms.

In summary, the SB transposon system has potential for gene delivery in Xenopus. This method simply requires classical injection of genetic materiel into fertilized eggs. SB procedure constitutes a useful complement of the REMI technique, particularly for the transgenesis of the sibling species X. tropicalis. This species with a diploid genome has become the genomic reference in amphibians and is therefore better suited for genetic approaches and transgenic studies.

Reported by

CNRS Research Department
UPR 3294, Université Paris-Sud, bât 445,IBAIC
91405 ORSAY
France
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