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Transgenesis is an essential, powerful tool for investigating gene function and the activities of enhancers, promoters, and transcription factors in the chromatin environment. In Xenopus, current methods generate germ-line transgenics by random insertion, often resulting in mosaicism, position-dependent variations in expression, and lab-to-lab differences in efficiency. We have developed and tested a Xenopus FLP-FRT recombinase-mediated transgenesis (X-FRMT) method. We demonstrate transgenesis of Xenopus laevis by FLP-catalyzed recombination of donor plasmid cassettes into F(1) tadpoles with host cassette transgenes. X-FRMT provides a new method for generating transgenic Xenopus. Once Xenopus lines harboring single host cassettes are generated, X-FRMT should allow for the targeting of transgenes to well-characterized integration site(s), requiring no more special reagents or training than that already common to most Xenopus labs.
Figure 3. Recombinase-mediated cassette exchange of the XOP → mCherry for the CarAct → eGFP transgene. D♀ eggs were in vitro fertilized with wild-type sperm and injected with donor DNA only (pf1XOP → mCherryf3) or with FLP cRNA. A: Eight weeks postfertilization the percent of eGFP-positive and mCherry-positive embryos were determined. B: Seven of 11 (64%) uninjected embryos tested were genotyped PCR-positive using eGFP-specific primers. C: Eleven of 12 (92%) embryos selected as eGFP-negative and fluorescent for mCherry in the eyes were genotyped PCR-positive using mCherry-specific primers (Note: very faint products were detected in F1 tadpoles 11 and 12). None of the mCherry-positive animals were found to contain the CarAct → eGFP transgene. C: control (no template); U1 and U2: gDNA from uninjected eGFP-positive and eGFP-negative F1 tadpoles, respectively.
Reproduced with permission of the Publisher, John Wiley & Sons
Figure 4. Cassettes as large as 4.5 kb can be exchanged in X-FRMT. A: J♀ F1 embryos were injected with FLP cRNA and donor DNA plasmids driving mCherry expression from the 549 bp XOP (Fig. 2) or 3,529 bp XSix6 promoter/enhancer (shown). B: After stage 45, the surviving embryos were scored for eGFP and/or mCherry expression. The number and percentage of surviving embryos as well as the total percentage of mCherry-positive embryos is indicated. C: The genomic DNA from J♀ F1 tadpoles grown from two uninjected (U1, U2) and two pf1XSix6 → mCherryf3 plasmid + FLP cRNA injected (T1, T2) embryos were analyzed by PCR. Primers specifically amplify the host (CarAct-F/eGFP-R1) or donor (Six6-F/mCherry-R2) transgenes (A and C).
Reproduced with permission of the Publisher, John Wiley & Sons
