利用Tol2转座子构建转基因草金鱼初探

Construction of Transgenic Goldfish Based on Tol2 Transposable Element

青鳉Tol2转座子是脊椎动物中发现的第一例天然具有活性的转座子,目前已经被成功应用于多种模式动物的转基因研究中。采用PCR的方法以质粒pCAgcGH为模板亚克隆出一段含有草鱼生长激素(GH)5个外显子、鲤鱼β-actin启动子及多个酶切位点的序列,并将这段序列与经双酶切处理的质粒pTol2-MCS-EGFP进行重组连接,得到重组质粒pTol2-GH-EGFP。采用显微注射的方法将重组质粒pTol2-GH-EGFP与体外合成的Tol2转座酶mRNA一起注入草金鱼受精卵中。通过观察绿色荧光和PCR检验绿色荧光蛋白和GH基因的表达,筛选出成功转入外源基因的草金鱼。阳性表达检出率为17.3%。利用Tol2转座子构建转基因草金鱼,不仅丰富了Tol2转座子的应用范围,而且为进一步利用Tol2转座元件进行观赏鱼转基因及基因表达研究奠定了基础。

Medaka Tol2 transposon （ Transposable element of Oryzias latipes, number 2 ） was the first natural active transposon in vertebrate, which had been successfully applied in transgenic research of variety model animals. The sequence containing five exons of grass carp growth hormone （ GH ） , carp beta-actin promoter and multiple restriction enzyme cutting sites was amplified based on pCAgcGH plasmid, and was inserted into pTol2-MCS-EGFP plasmid to construct recombinant pTol2-GH-EGFP plasmid. The recombinant pTol2-GH-EGFP plasmid and the transposase mRNA synthesized in vitro were coinjected into fertilized eggs of goldfish by microinjection. The transgenic goldfish were selected by observing green fluorescence and PCR, and expression rate was 17.3%. The construction of transgenic goldfish based on Tol2 transposable element, not only expanded the range of Tol2 transposon application, but also would be useful for studies of transgene and gene expression in ornamental fish.