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

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