The study on transgenic efficiency of PiggyBac transposon in the genome of Megalobrama amblycephala

Abstract:

PiggyBac(PB) transposon is derived from Trichoplusia ni of lepidopteran and has been widely used in transgenic and insertion mutagenesis studies in mouse. Currently, relevant research about PiggyBac transposons used in farmed fish has not yet been reported. To study insertion efficiency of PiggyBac transposon in the genome of M. amblycephala, we built pPBs-EF1α-eGFP donor plasmid with PiggyBac transposon left and right arms, EF1α promoter and eGFP gene,then co-injected with PiggyBac transposase mRNA into the 1-2 cell stage fertilized eggs of M. amblycephala. The concentrations of donor plasmid and transposase mRNA were 50 ng/μL and 100 ng/μL respectively. The eGFP fluorescence expression rate was 58.26%. In adult fish, PCR results demonstrated that integration efficiency of PiggyBac transposition system was 53.04% in M. amblycephala genome. Our data suggest that PiggyBac transposon can efficiently mediate gene insertion in M. amblycephala, which could be used in insertional mutagenesis in M. amblycephala.