Pan Fong Cheng, Chen Yonglong, Loeber Jana, Henningfeld Kristine, Pieler Tomas
Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany.
Dev Dyn. 2006 Jan;235(1):247-52. doi: 10.1002/dvdy.20608.
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.
已经描述了几种产生转基因青蛙的实验方法。在此，我们报告一种在非洲爪蟾中应用的新方法，该方法利用I-SceI巨核酸酶。这种核酸内切酶的特征在于它具有18bp的扩展识别位点，预计在7×10¹⁰bp的随机DNA序列中仅存在一次。将由两个I-SceI识别位点侧翼的各种报告基因构建体与I-SceI巨核酸酶一起注射到单细胞期的非洲爪蟾胚胎中。在优化条件下，我们观察到总体转基因频率达到10%或更高。注入的基因整合到基因组中并传递给F1代后代。Southern印迹分析表明，转基因整合了1至8个拷贝。因此，巨核酸酶辅助转基因为非洲爪蟾的转基因提供了一种简单且高效的工具。
