XB-ART-44188Genesis. March 1, 2012; 50 (3): 300-6.
Optimized transgenesis in Xenopus laevis/gilli isogenetic clones for immunological studies.
Xenopus laevis provides a unique animal model, alternative to mouse, to study immunology. Even though, several methodologies have been developed for the generation of transgenic Xenopus, to date none have been adapted for the X. laevis/gilli (LG) isogenetic clones that are essential for immunological studies. Since LG clones are generated via gynogenesis, transgenic methods using transgene integration into the sperm nuclei are not suited. Therefore, we have tested three alternative methods for LG transgenesis: the phiC31 integrase, the Sleeping Beauty transposase, and the I-SceI meganuclease. All three techniques produced transgenic LG clones; however, the I-SceI meganuclease was most effective. It resulted in high transgenesis efficiency (35-50%), bright nonmosaic GFP expression as well as stable germline transmission with 100% of the progeny carrying the transgene. Production of transgenic LG clones will allow us to modulate immune gene expression and further strengthen X. laevis as a biomedical model.
PubMed ID: 21954010
PMC ID: PMC3250570
Article link: Genesis.
Grant support: 1R03-HD061671-01 NICHD NIH HHS , R24-AI-059830-06 NIAID NIH HHS , T32-AI 07285 NIAID NIH HHS , R03 HD061671-02 NICHD NIH HHS , R24 AI059830-06 NIAID NIH HHS , T32 AI007285-24 NIAID NIH HHS , R24 AI059830 NIAID NIH HHS , R03 HD061671 NICHD NIH HHS , T32 AI007285 NIAID NIH HHS , R24-AI-059830-06 NIAID NIH HHS , T32-AI 07285 NIAID NIH HHS , 1R03-HD061671-01 NICHD NIH HHS , R03 HD061671-02 NICHD NIH HHS , R24 AI059830-06 NIAID NIH HHS , T32 AI007285-24 NIAID NIH HHS , R03 HD061671 NICHD NIH HHS , R24 AI059830 NIAID NIH HHS , T32 AI007285 NIAID NIH HHS
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|ge) and aneuploid (small) eggs. The small eggs were discarded while the diploid eggs were activated using UV-irradiated sperm. After activation, eggs were microinjected with the different constructs for transgenesis. Embryos were allowed to develop and were screened for GFP expression at stage 56 using a fluorescence stereomicroscope. Transgenic tadpoles were raised to adulthood to obtain F1 progeny. (b) Representative images of live nontransgenic (a) and transgenic larvae. Dejellied fertilized LG eggs were co-injected with (b) 1 ng of phiC31 integrase mRNA and 25 pg of CMV-GFP-DI-attB vector, (c) 0.1 ng of SB mRNA and 15 pg of HSV-GFP, (d) 10 I-SceI units and 80 pg of I-SceIFP in a total volume of 10 nl per egg. Reproduced with permission of the Publisher, John Wiley & Sons.|
|Figure 2. All eggs from GFP transgenic founder LG clones carry the transgene. Representative PCR of 28 unfertilized eggs from a GFP transgenic founder LG-6 frog (a) and 14 unfertilized eggs from a nontransgenic frog (b). GFP-specific primers (35 cycles) and EF1-α primers (control, 35 cycles) were used. Each lane represents a single egg. (NT) no template. Reproduced with permission of the Publisher, John Wiley & Sons.|
|Figure 3. Germline transmission of the GFP transgene to F1 LG progeny. F1 transgenic progeny were reared to adulthood and screened for GFP fluorescence. (a) Representative image of an adult GFP positive F1 LG transgenic clone. (b) Representative flow cytometry analysis of blood (including both red blood cells and lymphocytes) from two F1 transgenic LG clones (blue and red histograms) as well as a nontransgenic control frog (gray-shaded histogram). About 50,000 events were collected, gated on live cells, and analyzed for GFP expression using the FlowJo software. Reproduced with permission of the Publisher, John Wiley & Sons.|