John Antonius Verhees

Various expression-augmenting DNA elements benefit from STAR-Select, a novel high stringency selection system for protein expression.

The creation of highly productive mammalian cell lines often requires the screening of large numbers of clones, and even then expression levels are often low. Previously, we identified DNA elements, anti‐repressor or STAR elements, that increase protein expression levels. These positive effects of STAR elements are most apparent when stable clones are established under high selection stringency. We therefore developed a very high selection system, STAR‐Select, that allows the formation of few but highly productive clones. Here we compare the influence of STAR and other expression‐augmenting DNA elements on protein expression levels in CHO‐K1 cells. The comparison is done in the context of the often‐used cotransfection selection procedure and in the context of the STAR‐Select system. We show that STAR elements, as well as MAR elements induce the highest protein expression levels with both selection systems. Furthermore, in trans cotransfection of multiple copies of STAR and MAR elements also results in higher protein expression levels. However, highest expression levels are achieved with the STAR‐Select selection system, when STAR elements or MARs are incorporated in a single construct. Our results also show that the novel STAR‐Select selection system, which was developed in the context of STAR elements, is also very beneficial for the use of MAR elements.

Placing the RPL32 Promoter Upstream of a Second Promoter Results in a Strongly Increased Number of Stably Transfected Mammalian Cell Lines That Display High Protein Expression Levels

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.