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

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.

Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus

“…S3 ). While a few studies carried out electroporation on bivalve embryos ( 13 , 14 , 16 ), there is no report of electroporation on molluscan primary cells. Thus, we first tested whether scallop primary cells could be transfected by electroporation using luciferase-encoding mRNA and optimized basal electroporation conditions ( Fig.…”

“…The efforts from the 1990s to transfect bivalve cells have not been very successful because of the low efficiency of transfection methods and the lack of strong promoters ( 8 ). In bivalve cells and embryos, the promoters tested so far are 5′ upstream regions of Drosophila hsp70 ( 9 , 10 ), Crassostrea gigas actβ ( 11 ), ef1α ( 12 – 14 ), the long terminal repeats of Molony murine leukemia virus ( 9 , 15 ), and the cytomegalovirus immediate early (CMV IE) promoter ( 14 , 16 , 17 ), but practically reliable promoters have yet to be discovered. Recently, applications of genome-editing techniques are advancing in bivalve research ( 13 , 18 ).…”

Potential of genomic technologies to improve disease resistance in molluscan aquaculture

“…It may also be plausible to use sperm as the delivery vehicle for the CRISPR/Cas9 complex to edit the eggs after fertilization, an approach shown to be successful in chickens [126]. The abundance of active transposable elements in molluscan genomes could be adapted to allow insertion of exogenous DNA sequences, which has been successfully demonstrated in the pacific oyster to incorporate green fluorescing protein (GFP) [118]. Combined with a large number of gametes, low transgenesis and editing efficiency mean there is a clear need for a high-throughput screen to select for successfully edited/transgenic gametes/embryos.…”

The pond snail Lymnaea stagnalis

“…Transgenic snails have not yet been produced, although several techniques have been developed in mollusca but not yet used for functional assays [77][78][79]. The possibility to generate a knock-in snail line using the CRISPR technique was indicated for a sea water snail, where transient transgenic expression was achieved [80].…”