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• W3013116871 abstract "Achieving stable expression of a single transgene in mammalian cells remains challenging; even more challenging is obtaining simultaneous stable expression of multiple transgenes at reproducible, relative expression levels. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this BAC approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01- to 5-fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes. Second, we demonstrate little variation in expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making the choice of scaffolds more flexible. Third, we present a novel assembly scheme, BAC-MAGIC, for inserting multiple transgenes into scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved TG-EMBED toolkit, we simultaneously fluorescently labeled three nuclear compartments at reproducible, relative intensity levels in 94% of stable clones after a single transfection using a DHFR scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels." @default.
• W3013116871 created "2020-04-03" @default.
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• W3013116871 date "2020-03-27" @default.
• W3013116871 modified "2023-09-30" @default.
• W3013116871 title "Efficient and Reproducible Multigene Expression after Single-Step Transfection Using Improved BAC Transgenesis and Engineering Toolkit" @default.
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• W3013116871 doi "https://doi.org/10.1021/acssynbio.9b00457" @default.
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