PiggyBac Transposon-Mediated Transgenesis in the Pacific Oyster (Crassostrea gigas) - First Time in Mollusks.

Jun Chen,Xiaotong Wang,Zhuang Li,Baolu Zhang,Yongchuan Li,Zhongqiang Cai,Changlu Wu,Lei Wei,Wen Guo,Guangbin Li,Ting Guo

doi:10.3389/fphys.2018.00811

Abstract

As an effective method of transgenesis, the plasmid of PiggyBac transposon containing GFP (PiggyBac) transposon system has been widely used in various organisms but not yet in mollusks. In this work, piggyBac containing green fluorescent protein (GFP) was transferred into the Pacific oyster (Crassostrea gigas) by sperm-mediated gene transfer with or without electroporation. Fluorescent larvae were then observed and isolated under an inverted fluorescence microscope, and insertion of piggyBac was tested by polymerase chain reaction (PCR) using genomic DNA as template. Oyster larvae with green fluorescence were observed after transgenesis with or without electroporation, but electroporation increased the efficiency of sperm-mediated transgenesis. Subsequently, the recombinant piggyBac plasmid containing gGH (piggyBac-gGH) containing GFP and a growth hormone gene from orange-spotted grouper (gGH) was transferred into oysters using sperm mediation with electroporation, and fluorescent larvae were observed and isolated. The insertion of piggyBac-gGH was tested by PCR and genome walking analysis. PCR analysis indicated that piggyBac-gGH was transferred into oyster larvae; genome walking analysis further showed the detailed location where piggyBac-gGH was inserted in the oyster genome. This is the first time that piggyBac transposon-mediated transgenesis has been applied in mollusks.

Highlights

Many methods can be employed to achieve genetic transformation, including viral vectors, non-viral vectors and transposons
Studies have shown that the original piggyBac transposon has a broad host spectrum ranging from yeast to mammals, and this mobile element has been widely used for a variety of applications in a diverse range of organisms
We demonstrate that the piggyBac transposon system is suitable for Pacific oyster transgenesis

Summary

Introduction

Many methods can be employed to achieve genetic transformation, including viral vectors, non-viral vectors and transposons. PiggyBac transposon can introduce exogenous genes into the genome for constructing transgenic organisms or testing gene function (Yusa, 2015). PiggyBac transposons have been successfully used as a promising tool for basic genetic studies (Wang et al, 2008; Kaji et al, 2009; Woltjen et al, 2009; Yusa et al, 2009), gene therapies (Carlson et al, 2005) and construction of transgenic animals (Ding et al, 2005). Studies have shown that the original piggyBac transposon has a broad host spectrum ranging from yeast to mammals, and this mobile element has been widely used for a variety of applications in a diverse range of organisms. The piggyBac transposition system has not yet been utilized or characterized in mollusks

Results

Discussion

Conclusion