The Frog Prince:: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells
The Frog Prince:: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells
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DOI：
10.1093/nar/gkg910
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发表时间：
2003-12-01
影响因子：
14.9
通讯作者：
Ivics, Z
中科院分区：
文献类型：
--
作者：
Miskey, C;Izsvák, Z;Ivics, Z
从脊椎动物中分离的Tc 1/mariner转座因子超家族的成员由于其转座酶基因中突变的积累而转座失活。采用一种新的开放式阅读框架捕获法从蛙属物种尖音蛙（Rana pipiens）的基因组中分离不间断的转座酶编码区。分离的克隆与预期的非洲爪蟾转座酶基因序列相似性为90%，但含有一个未预期的相似于180 bp的区域，编码假定的转座酶的N-末端。没有发现这些天然基因是活跃的。因此，推导出转座酶基因的共有序列。这种工程转座酶和转座子反向重复序列共同构成了一种新型转座子系统的组成部分，我们将其命名为青蛙王子（FP）。FP与睡美人（Sleeping Beauty，SB）只有50%的序列相似性，在鱼类、两栖类和哺乳动物细胞系中催化高效的剪切粘贴转座。我们证明了使用FP转座在人类细胞中的高效基因捕获。FP是迄今为止描述的最有效的脊椎动物DNA转座子，并且在斑马鱼细胞中显示出比SB高70%的活性。青蛙王子可以极大地扩展我们在脊椎动物中进行遗传分析的可能性。
Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were similar to90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, similar to180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only similar to50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows similar to70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.