I-SceI meganuclease-mediated transgenesis in Xenopus.
Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.
Pan
Fong Cheng
FC
Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany.
Chen
Yonglong
Y
Loeber
Jana
J
Henningfeld
Kristine
K
Pieler
Tomas
T
D000818:Animals; D030801:Animals, Genetically Modified; D015139:Blotting, Southern; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D018014:Gene Transfer Techniques; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins; D019076:Transgenes; D014982:Xenopus laevis / Q000196:embryology
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific