Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

Abstract

16Achieving reproducible, stable, and high-level transgene expression in 17 mammalian cells remains problematic. Previously, we attained copy-number-18 dependent, chromosome-position-independent expression of reporter minigenes 19 by embedding them within a BAC containing the mouse Msh3-Dhfr locus (DHFR 20 BAC). Here we extend this "BAC TG-EMBED" approach. First, we report a 21 toolkit of endogenous promoters capable of driving transgene expression over a 22 novel gene circuits, involving the expression of multiple proteins, in many cases 61 at precise relative levels (25). While this approach has worked well in 62 prokaryotes and yeast, it has been difficult to implement in mammalian cells due 63 to the lack of suitable multi-transgene expression methods which overcome 64 chromosome position effects and allow expression of different transgenes at 65 reproducible relative levels. 66A commonly used approach to countering transgene silencing and 67 variegation has been through the inclusion of cis-elements. These include 68 insulators (26, 27), locus control regions (LCRs) (28, 29), scaffold/matrix 69 attachment regions (S/MARs) (30, 31), ubiquitous chromatin opening elements 70 (UCOEs) (32, 33) and anti-repressors (34); some of these regulatory elements 71have context-dependent and/or vector dependent activity. While these cis-72 elements improve transgene expression to varying degrees, they are insufficient 73 for chromosome-position independent, copy-number-dependent transgene 74 expression (29,(35)(36)(37). 75Additionally, in some transgene expression applications the ability to avoid 76 transgene chromosomal integration and eventually eliminate these transgenes 77 from the cells is highly desirable. Both viral-sequence based and non-viral, pEPI 78 based episomal vectors have been developed (38-41). Viral-based vectors have 79 the potential of causing transformation of the transfected cells (42), while pEPI-80 like vectors, containing a S/MAR sequence immediately downstream of an active 81 transcription unit, are mitotically stable without selection (43-47), and thus 82 cannot be removed from the cells. Moreover, transgenes on these episomal 83 vectors are still subject to silencing (48), possibly due to the prokaryotic or viral 84 sequences on these vectors (49, 50). 85Bacterial artificial chromosomes (BACs) carrying ~100-200 kb mammalian 86 genomic DNA inserts harbor most of the cis-regulatory sequences required for 87 expression of the endogenous genes contained within these genomic inserts. 88Previously we demonstrated how embedding minigene constructs at different 89 locations within the DHFR BAC provided reproducible expression of single or 90 multiple reporter genes independent of the chromosome integration site (51). 91Similar approaches were used by other labs for high-level recombinant protein 92 production (52, 53). Recently, our lab demonstrated stable transgene expression 93 after cell-cycle arrest or after terminal cell differentiation, using the BAC-TG 94 EMBED approach (54). All of these studies...

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

“…We found the presence of nonintegrated BAC transgenes propagating as episomes and their unequal segregation to be the source of this heterogeneity. The formation and propagation of BAC episomes are beyond the scope of the present study and are reported elsewhere . In the absence of continued drug selection, we would expect cells that have lost BAC transgenes will accumulate if there is any selective growth advantage for cells with fewer BAC copies.…”

“…The formation and propagation of BAC episomes are beyond the scope of the present study and are reported elsewhere. 50 In the absence of continued drug selection, we would expect cells that have lost BAC transgenes will accumulate if there is any selective growth advantage for cells with fewer BAC copies.…”