Abstract
The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1α, PGK, chicken β-actin and MC1) have been quantified and compared in mouse embryonic stem cells. To avoid the high variation in transgene expression which results from uncontrolled copy number and chromosomal position effects when using random insertion based transgenic approaches, we have adopted a PhiC31 integrase mediated cassette exchange method for the efficient insertion of transgenes at single copy within a defined and well characterized chromosomal position, ROSA26. This has enabled the direct comparison of constructs from within the same genomic context and allows a systematic and quantitative assessment of the strengths of the promoters in comparison with the endogenous ROSA26 promoter. The behavior of these exogenous promoters, when integrated at ROSA26 in both sense and antisense orientations, reveals a large variation in their levels of activity. In addition, a subset of promoters, EF1α, UbC and CAG, show an increased activity in the sense orientation as a consequence of integration. Transient transfection experiments confirmed these observations to reflect integration dependent effects and also revealed significant differences in the behaviour of these promoters when delivered transiently or stably. As well as providing an important reference which will facilitate the choice of an appropriate promoter to achieve the desired level of expression for a specific research question, this study also demonstrates the suitability of the cassette exchange methodology for the robust and reliable expression of multiple variant transgenes in ES cells.
Highlights
Stem cells and in particular embryonic stem (ES) cells are frequently used in gene function studies, where transgenes are introduced under the control of exogenous promoters
The constructs in their respective orientations were targeted to the ROSA26 locus via PhiC31 integrase mediated cassette exchange (Figure 1A), and 16 independent G418 resistant clones were isolated for each of these 18 different constructs
Multiple independent ES cell clones for each construct, integrated correctly at both the 59 and 39 ends, were identified and integration of a single copy of the transgenic construct was verified by Southern blot analysis
Summary
Stem cells and in particular embryonic stem (ES) cells are frequently used in gene function studies, where transgenes are introduced under the control of exogenous promoters. Literature which aids this choice is limited and existing comparisons of promoter strength are frequently contradictory or use methodologies which may be inappropriate for comparative studies. Gene targeting technologies in ES cells permit the insertion of transgenic constructs within defined genomic docking sites [1,2]. This refinement limits the high variability of expression which is frequently seen in models made by conventional pronuclear microinjection, where constructs are integrated at random and at varying copy number into the genome [3]. When comparisons between variant constructs are required, the targeted insertion of transgenic cassettes provides the most appropriate methodology, as the functional effects of construct variation can be assayed within a standardized genomic insertion site
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