A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells.
A comparison of exogenous promoter activity at the ROSA26 locus using a ΦiC31 integrase mediated cassette exchange approach in mouse ES cells.
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DOI：
10.1371/journal.pone.0023376
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Davies B
中科院分区：
文献类型：
--
作者：
Chen CM;Krohn J;Bhattacharya S;Davies B
对小鼠胚胎干细胞中9个普遍存在的启动子（ROSA26、CAG、CMV、CMVd1、UbC、EF1α、PGK、鸡β-actin和MC1）的活性进行了定量比较。为了避免在使用基于随机插入的转基因方法时由于拷贝数不受控制和染色体位置效应而导致的转基因表达的高度变异，我们采用了PhiC31整合酶介导的盒式交换方法，在一个明确且特征明确的染色体位置ROSA26内的单拷贝上高效地插入转基因。这使得能够直接比较相同基因组背景下的构建，并允许系统和定量地评估启动子与内源性ROSA26启动子的优势。当这些外源启动子以正义和反义取向整合在ROSA26上时，其行为显示出其活性水平的巨大变化。此外，EF1α、UbC和CAG等启动子的一个子集由于整合而在感觉取向上显示出增加的活性。瞬时转染实验证实了这些观察结果，反映了整合依赖效应，也揭示了这些启动子在瞬时或稳定传递时的行为存在显著差异。除了为选择合适的启动子以达到特定研究问题所需的表达水平提供重要参考外，本研究还证明了盒式交换方法在胚胎干细胞中健壮可靠地表达多变体转基因的适用性。
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.
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DOI：
10.1073/pnas.93.17.9067
发表时间：
1996-08-20
影响因子：
11.1
作者：
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