Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage
Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage
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DOI：
10.1073/pnas.97.21.11403
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发表时间：
2000-10-10
影响因子：
11.1
通讯作者：
Kawakami, N
中科院分区：
文献类型：
--
作者：
Kawakami, K;Shima, A;Kawakami, N
青鳉属鱼类Oryzias latipes的Tol 2元件属于转座子的hAT家族（流浪汉/Ac/Tam 3）。我们在这里报告的功能转座酶的Tol 2，能够催化其在斑马鱼的生殖系转座的鉴定。从Tol 2产生的转录本编码一个假定的转座酶，斑马鱼受精卵与mRNA转录体外共注射，使用Tol 2转录本的cDNA作为模板和携带突变体Tol 2的质粒DNA。其在推定的转座酶基因中具有缺失，但保留了必需的顺式序列。将注射的鱼饲养至成年，并与未注射的鱼交配，通过PCR和Southern杂交分析后代鱼的基因组DNA。从八条注射鱼中的一条获得的F-1鱼的一半在其基因组中含有Tol 2 DNA，但不含有载体部分。在这些F-1鱼，Tol 2插入在四个不同的位点被确定，和一些F-1鱼进行两个或三个不同的Tol 2插入，表明生殖系的创始人鱼是高度嵌合。测序分析显示，在所有情况下，Tol 2被斑马鱼基因组序列包围，并且在靶位点产生了8-bp的重复，表明Tol 2通过转座整合到斑马鱼基因组中。本研究从脊椎动物基因组中鉴定了一个基于DNA的转座因子的自主成员。因此，Tol 2转座子系统应用于开发新的转基因和插入突变的方法在斑马鱼和可能在其他鱼类。
The Tol2 element of the medaka fish Oryzias latipes belongs to the hAT family of transposons (hobo/Ac/Tam3). We report here identification of a functional transposase of Tol2 that is capable of catalyzing its transposition in the germ line of zebrafish Danio rerio. A transcript produced from Tol2 encodes a putative transposase, Zebrafish fertilized eggs were coinjected with mRNA transcribed in vitro, using cDNA of the Tol2 transcript as a template and a plasmid DNA harboring a mutant Tol2. which had a deletion in the putative transposase gene but retained necessary cis sequences. The injected fish were raised to adulthood and mated to noninjected fish, and genomic DNA of the progeny fish were analyzed by PCR and Southern hybridization. Half of F-1 fish obtained from one of eight injected fish contained the Tol2 DNA in their genomes but not the vector portion. Among these F-1 fish, Tol2 insertions at four different loci were identified, and some F-1 fish carried two or three different Tol2 insertions, indicating that the germ line of the founder fish is highly mosaic. Sequencing analyses revealed that, in all cases, Tol2 was surrounded by zebrafish genomic sequences, and an 8-bp duplication was created at the target site, indicating that Tol2 was integrated in the zebrafish genome through transposition. This study identifies an autonomous member of a DNA-based transposable element from a vertebrate genome. The Tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes.