Abstract
The use of high stringency selection systems commonly results in a strongly diminished number of stably transfected mammalian cell lines. Here we placed twelve different promoters upstream of an adjacent primary promoter and tested whether this might result in an increased number of colonies; this is in the context of a stringent selection system. We found that only the promoter of the human ribosomal protein, RPL32, induced a high number of colonies in CHO-DG44 cells. This phenomenon was observed when the RPL32 promoter was combined with the CMV, SV40, EF1-α, and the β-actin promoters. In addition, these colonies displayed high protein expression levels. The RPL32 promoter had to be functionally intact, since the deletion of a small region upstream of the transcription start site demolished its positive action. We conclude that adding the RPL32 promoter to an expression cassette in cis may be a powerful tool to augment gene expression levels.
Highlights
Several approaches have been described that positively influence gene expression levels
We tested whether a second promoter, placed upstream of the “primary” human β-actin promoter might have a positive influence on the number of colonies that are formed in the context of a very stringent selection system
Twelve promoters were chosen for testing: the viral CMV and SV40 promoters, the human β-actin, γ-actin [21, 22], ubiquitin C (UBC) [23], EF1-α [24], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [25], and five ribosomal protein (RPL32, RPLP1, RPS21, RPL8, and RPL41) promoters [26]
Summary
Several approaches have been described that positively influence gene expression levels. One typical approach is to flank a gene expression cassette with DNA elements that somehow augment gene expression levels Amongst these elements are MAR (Matrix Attachment Regions) elements [1,2,3], UCOEs (Ubiquitous Chromatin Opening Elements) [4, 5], insulators [6, 7], and STAR elements [8]. Without DNA elements such as STARs, Zeocin gene expression is not high enough and no colonies are formed as a result. This creates a very low background, which makes it easy to assess the (positive) influences of exogenous factors on the number of induced colonies. The addition of the heterologous RPL32 promoter to a gene expression cassette may be a powerful tool for augmenting gene expression levels
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