原创力文档a comparison of exogenous promoter activity at the rosa26 locus using a phic31 integrase mediated cassette exchange approach in mouse es cells比较rosa26外生子活动的轨迹使用phic31整合酶在小鼠胚胎干细胞介导盒式交换方法.pdfVIP
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a comparison of exogenous promoter activity at the rosa26 locus using a phic31 integrase mediated cassette exchange approach in mouse es cells比较rosa26外生子活动的轨迹使用phic31整合酶在小鼠胚胎干细胞介导盒式交换方法
A Comparison of Exogenous Promoter Activity at the
ROSA26 Locus Using a PhiC31 Integrase Mediated
Cassette Exchange Approach in Mouse ES Cells
1,2 1 1,2 1
Chiann-mun Chen , Jon Krohn , Shoumo Bhattacharya , Benjamin Davies *
1 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 2 Department of Cardiovascular Medicine, University of Oxford, Oxford,
United Kingdom
Abstract
The activities of nine ubiquitous promoters (ROSA26, CAG, CMV, CMVd1, UbC, EF1a, PGK, chicken b-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, EF1a, 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 diff
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