Efficient and reproducible multigene expression after single-step transfection using improved bac transgenesis and engineering toolkit

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 BAC "scaffold" containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this "BAC TG-EMBED" 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 BAC scaffolds more flexible. Third, we present a novel BAC assembly scheme, "BAC-MAGIC", for inserting multiple transgenes into BAC scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved BAC 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 BAC scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended BAC TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels.