piggyBac转座子介导的转基因叉尾斗鱼的构建

为探讨piggyBac转座子在鱼类动物中应用的可能性,以包含家蚕(Bombyx mori)肌动蛋白3启动子驱动的增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)基因的piggyBac质粒为载体,以及一个包含piggyBac转座酶的辅助质粒,采用显微注射的方法将其导入叉尾斗鱼(Macropodusopercularis)受精卵中,利用PCR技术证实了piggyBac转座子能够介导EGFP基因进入叉尾斗鱼基因组,并能够稳定遗传到下一代,符合孟德尔遗传规律。EGFP基因遗传到G1代的阳性鱼占交配鱼比率,即外源基因整合率为12.30%。实验证明,piggyBac质粒有可能成为水产动物转基因实验的新型载体。

Construction of Transgenic Macropodus opercularis by Transposon piggyBac

In order to study the feasibility of piggyBac transposon application in the fish, the plasmid consisting of the piggyBac inverted terminal repeats flanking a fusion of the Bombyx mori cytoplasmic actin gene BmA3 promoter and the enhanced green fluorescent protein (EGFP) gene and a nonautonomous helper plasmid encoding the piggyBac transposes was introduced into the zygote of the Macropodus opercularis through microinjection. The PCR identification indicated that the EGFP gene mediated by piggyBac transposon existed in the genome of the M. opercularis. Transgene was stably transferred to the next generation through normal Mendelian inheritance. The foreign DNA integration rate, i.e., the rate of number of G1 broods with EGFP positive fish to the number of fertile fish was 12.30%. These results prove that piggyBac plasmid can be a new vector for the transgenic experiment in fish. The system constructed in our experiment is feasible.