title: The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos.
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.
authors: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors: Luo-Dan
authors: LD
authors: Cheng
authors: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors: Xia-Yun
authors: XY
authors: Jiang
authors: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors: Yu-Mei
authors: YM
authors: Tian
authors: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China.
authors: Jie
authors: J
authors: Chen
authors: Key Laboratory of Freshwater Aquatic Genetic Resources, Shanghai Ocean University, Huchenghuan Road 999, Shanghai 201306, China. Electronic address: smzou@shou.edu.cn.
authors: Shu-Ming
authors: SM
authors: Zou
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000502:physiology*; D004625:Embryo, Nonmammalian; D006054:Goldfish / Q000235:genetics*; D054643:INDEL Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D019895:Transposases / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases
keywords: 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