PT  - JOURNAL ARTICLE
AU  - Binhui Zhao
AU  - Pankaj Chaturvedi
AU  - David L. Zimmerman
AU  - Andrew S. Belmont
TI  - Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC
AID  - 10.1101/708024
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 708024
4099  - http://biorxiv.org/content/early/2019/07/23/708024.short
4100  - http://biorxiv.org/content/early/2019/07/23/708024.full
AB  - Achieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC 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 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.