Ted H. J. Kwaks

Identification of anti-repressor elements that confer high and stable protein production in mammalian cells

The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number–dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.

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.