XB-ART-842Mech Dev February 1, 2006; 123 (2): 103-13.
Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease.
In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are ''half-transgenics'' suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis.
PubMed ID: 16413175
Article link: Mech Dev
Grant support: EY06675 NEI NIH HHS , EY10283 NEI NIH HHS , RR13221 NCRR NIH HHS
Genes referenced: pax6
Article Images: [+] show captions
|Fig. 1. Expression of the Pax6 reporter co-injected with I-SceI meganuclease into X. tropicalis embryos. (A) A reporter construct (IS-Pax6GFP) introduced into embryos shown in panels B–G, and I. The Pax6 promoter-GFP cassette is flanked at both ends by two I-Sce1 recognition sites. (B–D) GFP fluorescence observed in embryos co-injected with IS-Pax6GFP and I-SceI meganuclease (B, stage 20; C, stage 25; D, stage 41). (E) In situ hybridization analysis of GFP expression in a stage 26 embryo co-injected with IS-Pax6GFP and I-SceI meganuclease. The smooth expression in the eye and brain tissues and the spotty ectopic expression in ventral cells are indicated by arrows and arrowhead, respectively. (F,G) Putative half transgenic embryos showing GFP expression only in one side (F, GFP fluorescence at stage 41; G, in situ hybridization with GFP probe at stage 26). (H) GFP expression in stage 41 transgenic embryo generated by the REMI method using the same Pax6 promoter-GFP cassette. (I) Mosaic GFP expression in stage 41 embryo injected without I-SceI.|