As the generation of sexually mature Xenopus laevis
frogs takes about a year, we initially introduced the reporter gene and the Cre expression vector as stable transgenes simultaneously (Fig. ). Using this approach immediate evaluation of Cre recombinase action is possible in the founder generation. However, the number of healthy transgenic animals is limited and each individual will be unique as it has its own copy number and integration site of the transgene. Nevertheless, such double transgenic founder animals are potentially most valuable in experiments aimed at interfering with gene function. We envisage to select double transgenic founder animals with Cre-mediated activation of the cotransgenic fluorescence reporter in the most optimal fashion. These animals could then be crossed with animals containing a Cre-activated gene to interfere with gene function in the developing organism. This would lead to a more predictable outcome, as the Cre activity would already be defined and the cotransgenic reporter would be a most valuable marker to look for the functional consequence of the Cre-induced expression of the interfering gene. Based on the observation that different transgenes introduced simultaneously are frequently integrated at the same locus and show a coordinate expression (5
), we exclude a segregation of the Cre transgene and the reporter in the F1 generation. However, we cannot exclude that the Cre transgene is expressed in the germ line leading to the transmission of a recombined reporter.
Clearly, strains with separate reporter DNA and Cre-expressing transgenes have several advantages, too. It would allow the crossing in many combinations, an undertaking most promising in the long run, especially if the efforts of different laboratories are combined. This approach is also very attractive as it generates many animals with most similar phenotypes as the transgenes involved are integrated in identical copy number and at the same chromosomal locus between the various animals.
Analyzing five reporter females selected in the larval stage for its transgene expression, we obtained two females that transmitted a silent transgene. As the injection of Cre mRNA into the two-cell stage embryo results in an efficient activation of the reporter gene (Fig. D), we exclude the possibility that the transgene has been inactivated in a permanent way as, for example, by deletion of some crucial regulatory DNA element. In the three other transgenic reporter lines we obtained expression of the transgene in the offspring. Assuming one integration site, we would expect 50% of the F1 animals to be transgenic. Indeed, this was found in the larvae derived from Y2 (Table ) and all the non-expressing larvae lacked any transgene as assayed by PCR. But curiously, we observed only 30% blue fluorescent larvae in the F1 generation of female C5 (Table ). This unexpectedly low number may be due to a mosaic germ line of C5 or some uneven selection occurring during oogenesis that depends on differential transgene integration. Obviously, a detailed analysis of the F1 and F2 generation of the C5 founder female is required to sort out these various possibilities.
To rapidly score whether the reporter transgene can be targeted by Cre recombinase we injected Cre mRNA into two-cell stage embryos of the F1 generation. We observed in blue fluorescent larvae a mosaic pattern of yellow fluorescent myotomes. This partial effect possibly reflects the fact that the recombinase introduced as a mRNA into the two-cell stage embryo has a limited stability. We exclude the possibility that only a fraction of the cell nuclei can be targeted by the recombinase, as in the same strains we observed extensive muscle-specific recombination in crossing experiments (Table ). Injecting mRNA encoding Cre we also observed a silencing of the blue fluorescent protein expression without the appearance of yellow fluorescence. As frequently multiple copies are integrated into one locus in transgenic Xenopus
), we assume that the copy number of the reporter has been reduced by recombination without generating an active EYFP expression cassette. Possibly this transgene silencing is not observed in crossing experiments, as the continuous production of transgenic Cre will ultimately recombine all loxP sites and thus lead to an active EYFP gene. Most surprisingly, we detected upon Cre mRNA injection an induction of ECFP expression in non-fluorescent larvae. We speculate that in these non-fluorescent transgenic larvae a high copy number of the transgene leads to gene silencing and that Cre-mediated reduction of the copy number activates the transgene. Such a transgene activation upon Cre-mediated copy number reduction has been reported in transgenic mice (17
Our observation that the silent reporter in the females Y1 and C3 can be activated by Cre mRNA injection into the two-cell stage but not in crossing experiments using muscle-specific expressed Cre may indicate that the silent transgene can only be activated early in embryogenesis. Thus, this perplexing effect does not interfere in crossing experiments.
Tail regeneration in amphibians is an easy model to address the question how blastema formation occurs and how the fully differentiated regenerate arises (15
). In the axolotl (Ambystoma mexicanum
) it has been established that muscle fibers injured by amputation are dedifferentiated to form mononucleated cells that populate the newly formed blastema (18
). By fluorescence labeling of single muscle fibers it has been estimated that up to 30% of the cells in the blastema are derived from mature muscle cells. As in this analysis the fluorescent label just marked the cell content, it was not possible to analyze whether the dedifferentiated muscle cell nuclei would redifferentiate into muscle cells or also transdifferentiate into non-muscle cells. In our approach muscle cells are irreversibly marked due to the Cre-mediated activation of the EYFP gene. Therefore, we conclude from the absence of any non-muscle cells with yellow fluorescence that transdifferentiation of muscle cells is not a major event in tail regeneration.
In general, at early stages of tail regeneration we observed an extensive reactivation of transgene expression at the site of amputation. We assume that this reflects the proliferative stimulus acting on the transgenic CMV promoter whose activity is known to be sensitive to proliferative stimuli in transgenic mice (19
). In transgenic larvae containing Cre-induced yellow muscle, transgene activation at the site of tail amputation involves predominantly the expression of blue fluorescent protein in the multinucleate myotome. This indicates a preferential transcriptional activation of nuclei with unrecombined transgene. It is not clear whether this represents myotomes that are not yet fully differentiated or whether nuclei with unrecombined transgene are preferentially activated within a myotome. Consistent with the finding that blue fluorescence predominates at the site of amputation we detect blue fluorescence in the newly formed blastema and this is maintained throughout the outgrowth of the regenerating tail. Only at later stages of development can yellow fluorescence be detected in the myotomes.