Cytoplasmic Microinjection of <i>piggyBac</i> Transposase mRNA and Transposon Vectors for Efficient <i>In Vitro</i> Production of Transgenic Porcine Parthenotes

Abstract

The efficient production of transgenic (Tg) piglets has remained a challenge in the field of domestic animal studies. Unlike mice, the pronuclei of pig zygotes cannot be easily studied because of the abundance of lipid droplets. Therefore, the zygotes must be briefly centrifuged before pronuclear injection (PNI) to move the lipid droplets to the periphery of the zygote for PNI-mediated production of Tg piglets. However, this procedure is temporal because lipid droplets return to the original space during PNI, hampering the consecutive PNI. Cytoplasmic injection (CPI) of nucleic acids is comparatively simple than PNI because CPI does not require such pre-centrifugation. Unfortunately, CPI using purified DNA fragments is inadequate for creating Tg piglets because it is challenging to integrate nucleic acids into the host genome. <em>PiggyBac</em> (PB), one of the transposons, is a valuable tool enabling efficient chromosomal integration of a transgene. The PB-mediated gene transfer requires two components, namely, transposase and transposons harboring the gene of interest (GOI) flanked by PB acceptor sites. We speculate that the CPI of transposase mRNA and transposons could accelerate the chromosomal integration of the GOI in pig zygotes. To prove this hypothesis, we performed CPI using transposase mRNA (super PB transposase mRNA) + transposon DNA carrying the enhanced green fluorescent protein (<em>EGFP</em>) cDNA (referred to as “pT-EGFP”), transposase expression plasmid DNA (referred to as “pTrans”) + pT-EGFP, pT-EGFP alone, or non-transposon EGFP expression plasmid DNA using porcine parthenotes. Consequently, 50% (2/4 tested) of green-fluorescent embryos exhibited chromosomal integration of GOI. In contrast, green-fluorescent embryos derived from CPI with pTrans + pT-EGFP or pT-EGFP alone did not show chromosomal integration. We used mRNA for super PB transposase, which is an engineered hyperactive version of the wild-type PB transposase. To conclude, the PB system, based on the CPI of super PB transposase mRNA + transposon DNA, could be useful for producing Tg porcine parthenotes.