title: 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.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Bian
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Belmont
mesh_terms: D000818:Animals; D002843:Chromatin / Q000737:chemistry; D055012:Chromosomal Position Effects; D022202:Chromosomes, Artificial, Bacterial; D033481:Chromosomes, Mammalian; D018628:Gene Dosage; D015870:Gene Expression; D017930:Genes, Reporter; D051379:Mice; D041681:NIH 3T3 Cells; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D019076:Transgenes
chemical_list: D002843:Chromatin; D013762:Tetrahydrofolate Dehydrogenase
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