While these cis-elements improve transgene expression to varying degrees, they are insufficient for chromosome-position independent, copy-number-dependent transgene expression ( 29, 35– 37).Īdditionally, in some transgene expression applications the ability to avoid transgene chromosomal integration and eventually eliminate these transgenes from the cells is highly desirable. These include insulators ( 26, 27), locus control regions (LCRs) ( 28, 29), scaffold/matrix attachment regions (S/MARs) ( 30, 31), ubiquitous chromatin opening elements (UCOEs) ( 32, 33) and anti-repressors ( 34) some of these regulatory elements have context-dependent and/or vector dependent activity. ![]() While this approach has worked well in prokaryotes and yeast, it has been difficult to implement in mammalian cells due to the lack of suitable multi-transgene expression methods which overcome chromosome position effects and allow expression of different transgenes at reproducible relative levels.Ī commonly used approach to countering transgene silencing and variegation has been through the inclusion of cis-elements. For example, a common application in the emerging field of synthetic biology is the design of novel gene circuits, involving the expression of multiple proteins, in many cases at precise relative levels ( 25). Such limitations are compounded when the simultaneous and reproducible expression of multiple transgenes is required. Together these transgene silencing mechanisms result in unpredictable transgene expression levels that do not correlate with copy number and are unstable with long-term culture or changes in the cell physiological or differentiated state ( 22– 24). Moreover, foreign sequences by themselves are targets for epigenetic silencing ( 16– 19), and transgene concatamers can induce the formation of heterochromatin ( 20, 21). ![]() Plasmid-, lentivirus- and transposon-based systems, all still show varying degrees of chromosome position effects ( 9, 10) and position effect variegation (PEV) ( 11– 15). Examples of such applications include the expression of multiple fluorescent proteins for live-cell imaging ( 6), the expression of the four or more Yamanaka transcription factors for efficient generation of induced pluripotent stem (iPS) cells ( 7), and the expression of multiple proteins for reconstitution of protein complexes ( 8).ĭespite the currently widespread use of transgene expression, most transgene expression systems still suffer from serious experimental limitations. Applications of transgene expression range from the elucidation of gene function by ectopic expression of selected transgenes, to the expression of transgenes for gene therapy, and to the overexpression of genes for production of biopharmaceuticals ( 1– 5). Transgene expression has been widely used in both basic research and biotechnology. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. ![]() Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Here we extend this “BAC TG-EMBED” approach. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouse Msh3- Dhfr locus (DHFR BAC). Comments: I am a new user of the application but it has given me good results, it is a bit slow but have realized that using it more often I have managed to move faster.Achieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic.
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