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Strategy for selective acquisition of transgenic marmosets using the piggyBac transposon system

Transgenic (Tg) nonhuman primates (NHPs) are excellent potential models due to their physiological similarities with humans. In NHPs, the lentiviral vector is the only available transgenesis method, but transgene sizes are limited. The piggyBac (PB) transposon system is a DNA element sandwiched between two terminal inverted repeats (TRs) that allows transposases to introduce larger genes than lentiviral vectors. However, embryos with integrated transgenes are difficult to distinguish using this system. To address this issue, we placed the Enhanced Green Fluorescent Protein (EGFP) gene inside the TRs and the humanized kusabira orange 1 (hKO1) fluorescent protein gene with the proteolysis-promoting sequence d2PEST outside the TRs to create an indicator for gene integration using fluorescent protein expression. In HEK293T cells, hKO1 and EGFP co-expression after transfection gradually changed to decay of hKO1 expression and enhancement of EGFP expression by transposase within five days. In mouse and marmoset embryos, hKO1 expression was attenuated, while only EGFP expression was observed at the blastocyst stage. All offspring obtained through embryo transfer of EGFP-expressing embryos were Tg; thus, we established a new PB system capable of determining Tg embryos and successfully produced Tg marmosets, for the first time, in NHPs.