Efficient and Reproducible Multigene Expression after Single-Step Transfection Using Improved BAC Transgenesis and Engineering Toolkit

Abstract

Achieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-numberdependent, 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.

The Regenerative Power of Stem Cells: Treating Bleomycin-Induced Lung Fibrosis

“…Bacterial artificial chromosome (BAC)-mediated stable gene expression is a powerful tool that enables the introduction of large genomic regions, including entire genes and their regulatory elements, into cells to achieve stable and physiologically relevant gene expression. 126 , 127 This technique can be particularly useful in the context of lung fibrosis for introducing key reporters for lineage tracing, or through controlled expression of genes or signaling pathways regulating the differentiation and function of alveolar progenitor cells, including AT2 cells.…”