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.
Ogino
Hajime
H
Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.
McConnell
William B
WB
Grainger
Robert M
RM
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018014:Gene Transfer Techniques; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes / Q000235:genetics; D014981:Xenopus / Q000235:genetics*; D014982:Xenopus laevis / Q000235:genetics; D015053:Zygote / Q000637:transplantation
D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific