Large BACs transfect more efficiently in circular topology

The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells. [Display omitted] •The size and topology of DNA can influence transfection of mammalian cells.•100 kb circular BAC transfected almost 9-fold more efficiently than linear BAC.•BAC topology had minimal influence on transgene expression in eGFP-positive cells.