关键词： Autonomous excision Goldfish Tgf2 transposon ORF PCR Tc1 Tgf2 Tol2 Transposase Zebrafish aa amino acid(s) base pair(s) bp gfTP goldfish transposase hAT hobo of Drosophila, Ac of maize and Tam3 of snapdragon kb kilobase(s) or 1000bp mRNA message RNA open reading frame pCS2 plasmid containing CMV and SP6 promoter polymerase chain reaction transposon of Caenorhabditis elegans transposon of Oryzias latipes transposon of goldfish

Mesh ： Animals Animals, Genetically Modified Base Sequence DNA Transposable Elements / physiology Embryo, Nonmammalian Goldfish / genetics INDEL Mutation / genetics Plasmids / genetics Transposases / genetics Zebrafish / embryology genetics

来  源：   DOI：10.1016/j.gene.2013.11.084   PDF(Sci-hub)

Abstract：
The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300-500bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon.
摘要：
金鱼(Carassiusauratus)Tgf2转座子是属于hAT转座子家族的脊椎动物DNA转座子。在这项研究中,我们构建了含有全长Tgf2转座子（pTgf2质粒）或部分缺失的Tgf2转座子（ΔpTgf2质粒）的质粒，并在一到两个细胞阶段将这些质粒微注射到受精的斑马鱼（Daniorerio）卵中。通过PCR分析从胚胎中提取的DNA以评估瞬时切除，如果有的话,并验证Tgf2是否为自主转座子。结果表明，在注射ΔpTgf2质粒的胚胎中未检测到切除特异性条带，在注射pTgf2的胚胎中检测到300-500bp的条带，这表明全长含Tgf2的质粒可以在斑马鱼胚胎中进行自主切除。从注射pTgf2的24个胚胎中克隆的DNA进行测序，结果表明，Tgf2在斑马鱼胚胎中进行了自我切除。从与ΔpTgf2和体外转录的转座酶mRNA共注射的胚胎中提取的DNA的克隆和PCR分析表明，部分缺失的含Tgf2的ΔpTgf2质粒也进行了切除，在功能性转座酶mRNA的存在下。从25个与ΔpTgf2共同注射的胚胎中克隆的DNA和转座酶mRNA进行测序，结果表明部分缺失的Tgf2转座子质粒被切除。这些结果表明，Tgf2转座子的切除是由Tgf2转座酶介导的，这反过来证实Tgf2是一个自主转座子。
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