Methods to create a stringent selection system for mammalian cell lines

Abstract

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8–131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8–14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines.

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Cell-type-specific profiling of protein–DNA interactions without cell isolation using targeted DamID with next-generation sequencing

“…The frequency of translational initiation at the secondary ORF is inversely proportional to the length of the primary ORF, allowing very fine control over protein translation levels. 1,5,6 We have shown that TaDa fusion proteins can be driven in the Drosophila CNS throughout development and adult life without toxicity.…”

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…The inefficient rate of IRESmediated translation initiation of the short peptide combined with the necessity for subsequent downstream re-initiation of translation (post-IRES) at the start codon of the selection marker theoretically ensures that only those clones with very high levels of transcription and translation of this entire construct can survive. This system allows the fine tuning of the selection strength since by increasing the length of the short peptide the rate of translation of the selection marker ORF is reduced (Van Blokland et al 2011). As expected, this system produces fewer producer clones and a lower proportion of non-producers, but those that survive have high levels of production.…”

“…Although in the authors' experience, poor translation efficiency results in lower product concentration on average, especially for recombinant monoclonal antibodies (Lonza Biologics, unpublished data). Further development of a stringent selection system incorporating an IRES has been described by Van Blokland et al (2011). In this work the encoded multicistronic mRNA consists of an upstream product gene and a downstream (mutant) zeocin selection marker which are physically linked by both a single IRES and an intervening sequence encoding for a short peptide.…”