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Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping [single-cell RNA-seq]

ABSTRACT: Pronuclear microinjection is the most widely used method for producing transgenic (Tg) animals. Because its efficiency is typically <20%, phenotypic characterization of Tg animals is usually performed from the next generation (F1) onwards. 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 founder (F0) embryos are transgenic. 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. Our findings establish a highly efficient piggyBac-based platform that expands experimental options for Tg animal production by supporting rapid F0-based phenotypic assessment while also facilitating efficient generation of F0 animals for subsequent breeding.

ORGANISM(S): Mus musculus

PROVIDER: GSE306203 | GEO | 2026/06/11

REPOSITORIES: GEO

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Highly efficient and low-mosaicism piggyBac transgenesis platform for rapid founder phenotyping [Amplicon-seq]

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