BAC transgene arrays as a model system for studying large-scale chromatin structure

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. This method is extendable to reproducible, high level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes. Finally, we show that the removal of a divergent promoter region from the DHFR BAC results in ~10 folds increase of reporter mini-gene 18 expression level. We suggest the BAC TG-EMBED should be useful in a wide range of applications involving transgene expression. INTRODUCTION Plasmid based expression cassettes using cDNA mini-genes driven by viral or cloned eukaryotic promoters represent the most common method for expression of transgenes. Stable expression is usually achieved by integration of these cassettes into the host eukaryotic genome. However, expression levels of these mini-genes are typically greatly influenced by the chromatin structure surrounding the integration site, producing chromosome position effects, sometimes accompanied by variegation of expression(Karpen, 1994). Transgenes integrated into repressive chromatin regions are expressed at low levels and tend to be silenced over time. This effect is particularly pronounced in mammalian cells. A second phenomenon contributing to low levels of transgene expression is multi-copy transgene silencing, observed for most plasmid mini-genes(Dorer and Henikoff, 1997; Garrick et al., 1998). In transgene silencing, expression per gene copy tends to decrease with increasing transgene copy number such that transgene expression levels do not increase proportionally with copy number and very high copy number insertions may express at levels comparable or even lower than single copy insertions. The combined impact of chromosome position effects and transgene silencing makes typical transgene expression in mammalian cells both unpredictable and unstable, and has hindered both industrial and clinical applications as well as biomedical research applications. These problems are compounded when cell lines expressing multiple transgenes are required. As just one example, a number of recombinant proteins are important therapeutic 19 reagents with enormous market value. Mammalian cell culture has been the dominant expression system for therapeutic protein production as it facilitates both proper protein folding and posttranslational modifications(Andersen and Krummen, 2002; Wurm, 2004). In the absence, however, of a robust, single-step method for reliable, high-level, multi-copy transgene expression, gene amplification remains the method of choice for obtaining high expressing cell clones(Gandor et al., 1995). This process of gene amplification, in which cell mutants carrying hundred of copies of an inserted mini-gene are gradually selected, requires repeated rounds of cell selection, subcloning, and clone characterization over a period of many months. Even then, selection of amplified cell clones with high level, stable expression can be difficult and unpredictable, in many cases requiring a year or more for clone development and stabilization. To improve the efficiency and minimize the unpredictability of transgene expression, various cis-regulatory elements have been used to flank transgenes to maintain accessible chromatin structure and counteract chromosome position effects. Locus control regions (LCRs)(Grosveld et al., 1987), insulators(Chung et al., 1993; Pikaart et al., 1998), ubiquitous chromatin opening elements (UCOEs)(Antoniou et al., 2003; Williams et al., 2005), Scaffold/Matrix associated regions (SAR/MARs)(Kim et al., 2004; Zahn-Zabal et al., 2001), and antirepressor elements (STAR) (Kwaks et al., 2003)have all been shown to improve transgene expression to some degree(Kwaks and Otte, 2006). Locus control regions were identified as DNA sequences conferring copy number dependent, position independent expression close to endogenous gene expressions in transgenic mice(Li et al., 2002). Specifically, this property of copy number dependent expression is used as an operational definition distinguishing LCR 20 elements from enhancers. Known LCRs generally function in a tissue-specific manner, restricting their use to certain cell types. Also, as demonstrated for the beta-globin LCR, they are thought to act by interacting with only one promoter at any one moment in time, limiting their use to promote the expression of multiple transgenes simultaneously(Wijgerde et al., 1995). A “mini-locus” cosmid construct piecing together the LCR, 3’ beta-globin DNase I hypersensitivity site, and other beta-globin regulatory sequences showed highly linear, copy number dependent beta-globin expression over a 1-94 range in cosmid copy number(Grosveld et al., 1987). Copy number dependent expression has also been reproduced after random insertion of 244 kb or 155 kb yeast artificial chromosomes containing the intact beta-globin locus(Peterson et al., 1998). Instead, a 100 kb DNA genomic DNA region containing the entire beta-globin locus and LCR on a bacterial artificial chromosome (BAC) produced variable position effects in transgenic mice, with BAC transgenes expressing anywhere from 0-105% the level of the endogenous genes, with most lines expressing at ~1/3 the level of the endogenous genes(Kaufman et al., 1999). To reduce the original cosmid mini-LCR to a size allowing the use of more traditional cloning vectors, including viruses, “micro-LCRs” have been constructed using various combinations of the small DNA regions corresponding to the DNase I hypersensitivity sites of the LCR. These micro-LCRs are necessary but not sufficient for conferring chromosome position independent expression, with additional and different regulatory regions present in the introns and intergenic regions of specific globin genes, possibly arranged in a specific spatial pattern, required to confer copy number dependent expression(Bharadwaj et al., 2003; Guy et al., 1996; Kang et al., 2004; Rubin et al., 2000; Stamatoyannopoulos et al., 1997). For all of these reasons, use of LCRs in cloning vectors as a general tool for 21 conferring high-level protein expression or multi-gene transgenesis may be limited. Insulators are another important class of cis-regulatory elements used to shield transgenes from chromosome position effects created by spreading of activating or repressive chromatin marks from regions flanking the transgene insertion site(Chung et al., 1997). Most published studies using insulator sequences have not included careful analysis of the degree to which these sequences confer copy number dependent expression(Chung et al., 1997; Sekkali et al., 2008). In at least one case where such analysis was done, the chicken HS4 insulator was not sufficient to confer copy number dependent transgene expression in B cells(Truffinet et al., 2005). Copy number dependent expression in this system required additional, LCR-like cis regulatory sequences in the transgene together with the insulator sequence. Most of the other cis-regulatory elements described above also are not sufficient to confer both position independent and copy number dependent transgene expression. Here we describe a new transgene expression system based on embedding mini-gene constructs within large, cloned mammalian genomic DNA regions. When these DNA constructs are integrated into the mammalian genome they appear to create a reproducible, favorable chromatin environment for transcription independent of their chromosome integration site. We have implemented this system using bacterial artificial chromosomes (BACs) as the cloning vector. Using GFP and mRFP reporter mini-gene constructs, we show that by inserting these reporter genes into a BAC containing the mouse DHFR locus we obtain position independent, copy number dependent expression of reporters in cells clones carrying one to several hundred copies of the BAC. This contrasts with the typical position dependent, copy number independent, and lower expression values observed after directly transfecting cells with the very same mini-genes, with or without flanking chicken HS4 core 22 insulator sequences. Therefore in a single stable transformation we can isolate cell clones expressing mini-genes amplified up to several hundred folds. These cell clones show stable chromosome karyotype, transgene copy number, and expression over many passages in the presence of selection. Moreover, we also demonstrate position independent, copy number dependent, simultaneous expression of both RFP and GFP reporter genes inserted within a single BAC construct, showing the ability to use this method for multi-gene transgenesis. This BAC TG-EMBED (BAC TransGene EMBEDded) method provides a new methodology for single and multi-transgene expression capable of facilitating a wide range of applications.