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Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED

Gene Therapy volume 25, pages 376–391 (2018)Cite this article

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Abstract

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the “BAC TG-EMBED” method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.

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Acknowledgements

This work was supported by grant R01 GM098319 from the National Institute of General Medical Sciences (ASB). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank Peter Jones (University of Nevada Medical School) for providing pCpGvitro-neo-ZGFP and T. Brennan (University of California, San Francisco, San Francisco, CA) for FUGW plasmids. We also thank A. Smith (University of Cambridge, Cambridge, England, UK) for HM1 ES cells. We are also grateful to Edith Heard (Curie Institute) for providing Dhfr BAC (clone 057L22 from CITB mouse library).

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This work is NIH funded.

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  1. David L. Zimmerman

    Present address: Biology Department, College of the Ozarks, Point Lookout, MO, USA

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  1. Department of Cell and Developmental Biology, University of Illinois, Urbana, IL, USA

    Pankaj Chaturvedi, Binhui Zhao, David L. Zimmerman & Andrew S. Belmont

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Chaturvedi, P., Zhao, B., Zimmerman, D.L. et al. Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED. Gene Ther 25, 376–391 (2018). https://doi.org/10.1038/s41434-018-0021-z

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