BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression

Abstract

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.

Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice

“…This selection may partly explain the relatively limited antiviral capacities of the transgenic mouse lines. As primary cell selection and nuclear transfer is the principle technique of breeding large transgenic animals, locus control regions (LCR) [39] or artificial chromosomes [40] could be employed as position-independent delivery methods to elicit more efficient RNAi expression and to reduce the cost of selecting effective offspring.…”

3.4 Rational Approaches for Transgene Expression: Targeted Integration and Episomal Maintenance

“…One of the reasons might be that the nature of the preferred locus of transgene expression is not yet identified. However, proof of principle has been shown for the well-characterized Rosa26 locus identified in the mouse genome [239,240] or the dhfr locus [241].…”

Recent advances in recombinant protein production

“…Therefore, BAC-based vectors confer copy-number dependent and chromatin position independent expression, making BACs very attractive candidates for expression vectors. Indeed, due to their characteristics, BAC-based expression vectors have been vastly used during the past 15 years in the transgenic mouse field; 19 surprisingly, only a few examples have been published using BACs as expression vectors applied to recombinant protein production in mammalian cells 20 - 23 …”

“…Furthermore, our results have been elegantly confirmed by other researchers. A 175 kb BAC-based expression vector containing the dihydrofolate reductase ( Dhfr ) resulted in copy number-dependent and stable expression in NIH 3T3 cells 20 . Thus, other BAC-based expression vectors containing other loci than the Rosa26 can also be successfully used to establish cell lines.…”