Transpososition of Tol-2 element by means of transposae mRNA injection into medakafish eggs

我们已经确定了一个新的终端反向重复类转座因子青鳉鱼。该元件被设计为Tol-2。通过PCR分析含有Tol-2拷贝的基因组区域来检测胚胎发生期间Tol-2的切除。Tol-2全长4681 bp。Tol-2含有4个ORF，其氨基酸序列与玉米的Ac、果蝇的流浪汉和金鱼草的Tam 3相似，均为活性转座因子。因此，Tol-2是建立鱼类基因标签系统的独特材料。我们开发了一种新的瞬时胚胎切除试验中，青鳉受精卵共注射mRNA转录在体外使用的Tol-2 cDNA作为模板和质粒DNA携带Tol-2元件内的b-半乳糖苷酶基因。只有当与Tol-2 mRNA共注射时，Tol-2元件才能被有效地切除。通过使用含有Tol-2的质粒载体有效地测量切除活性。

We have identified a novel terminal-inverted-repeat class transposable element in the medaka fish Oryzias latipes. The element was designed Tol-2. Excision of Tol-2 during embryogenesis was detected by a PCR analysis of a genomic region containing a Tol-2 copy. Tol-2 is 4681 bp in length. Tol-2 contains 4 ORFs which have amino acid sequence similarity with Ac of maize, hobo of Drosophia and Tam3 of snapdragon, which are all active transposable elements. Tol-2 is thus a unique material for establishing a gene tagging system in fish. We developed a novel transient embryonic excision assay in which medaka fertilized eggs were coinjected with mRNA transcribed in vitro using the Tol-2 cDNA as a template and a plasmidDNA harboring a Tol-2 element inside the b-galactosidease gene. The Tol-2 element could be efficiently excised only when co-injected with the Tol-2 mRNA. The excision activity is measured efficiently by using the Tol-2 containing plasmid vector.