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. 2023 Jul 14;14(7):431. doi: 10.1038/s41419-023-05951-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A piggyBac transposon-based gene search vector (PB-GSV) contains TRE (tetracycline response element) and CMV-Neo-PA (neo-expression cassette). PB-GSV could be efficiently integrated into the genome when co-transfected with transposase mPB. Tetracycline transactivator (tTA) could initiate mutagenesis when binding to the TRE element. This mutagenic effect was turned off in the presence of tetracycline (+Dox) (Tet-off system). The piggyBac-based mutagenic units combined with the Tet-off system resulted in a regulable high-throughput mutagenic system. B Different mutagenic effects were initiated by the sense RNA or antisense RNA depending on the integration orientation of PB-GSV in the genome. C Flow chart illustrates the screening including the establishment of mutagenic libraries, screening metastatic lesions in mouse models, functional validation of the metastatic ability of the subpopulations cultured from the metastatic lesions in the first-round screening in vivo by turning off the mutagenesis through the Tet-off system. The metastatic lesions in the second-round screening in vivo were cultured, and the integration sites were analyzed. D Titration of the transfection efficiency of the gene search vector. E High integration efficacy was achieved with an optimized ratio of PB-GSV and mPB. F The integration efficacy was represented by the number of G418-resistant clones stained by methylene blue.