Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping

Although pronuclear microinjection is the most widely used method for producing transgenic (Tg) animals, phenotypic characterization is usually performed from the next generation onwards because the production efficiency is limited. Conventional Cre-loxP-based conditional knockout (cKO) mouse production requires generating two genetically modified strains and multiple rounds of breeding before cKO mice are available for analysis. Here, we optimized a piggyBac transposon-based method of Tg mouse production and established conditions under which nearly all F₀ embryos are Tg. Using a single-cell RNA sequencing-based strategy, we characterized mosaicism in F₀ embryos and demonstrated that piggyBac-mediated transgene integration occurs early in embryonic development. We also achieved ∼70% efficiency in generating bacterial-artificial-chromosome-Tg mice. By combining this method with genome editing, we developed a strategy for tissue-specific-knockout phenotyping in the F₀ generation. This platform expands experimental options for Tg animal production by supporting rapid F0-based phenotypic assessment and efficient founder generation for subsequent breeding.