Construction of I-SceI transgenic elements and assessment of its transgenic efficiency in zebrafish

Abstract:

Meganuclease I-SceI is an endonuclease, which is derived from the intron of mitochondrial 21S rRNA gene of Saccharomyces cerevisiae and has been applied to transgenic study in recent years. Usually, to use this system, target plasmid and I-SceI protein were microinjected together. However, preparation of the I-SceI protein for microinjection is laborious and time consuming. Replacing the protein by I-SceI mRNA would be more convenient. In this study, the transgene of fluorescence protein was mediated by I-SceI mRNA to assess its transgenic efficiency in zebrafish. First, helper plasmid with I-SceI coding sequence pCS2-I-SceI was constructed and I-SceI mRNA was transcripted in vitro. Donor plasmid, pI-SceI-EF1α-eGFP and pI-SceI-EF1α-RFP donor plasmid with I-SceI recognition sites, EF1α promoter and eGFP(RFP) gene were constructed, and co-injected with I-SceI mRNA into the 1-2 cell stage fertilized eggs of Danio rerio. The concentrations of donor plasmid and I-SceI mRNA were 50 ng/μL and 100 ng/μL respectively. The eGFP fluorescence expression rate was 75.86% in 48 h. The fluorescence can be observed in the head, body and tail of zebrafish and became stronger and stronger with the development of embryos. Our data suggest that I-SceI can efficiently mediate gene transfer and has the potential to apply in the transgene of economic fish.