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.