caa a22 4500 445823267 CHVBK 20180317145251.0 cr unu---uuuuu 170323e20110801xx s 000 0 eng 10.1007/s10616-011-9354-9 doi (NATIONALLICENCE)springer-10.1007/s10616-011-9354-9 Methods to create a stringent selection system for mammalian cell lines [Elektronische Daten] [H. Van Blokland, F. Hoeksema, M. Siep, A. Otte, J. Verhees] 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. The Author(s), 2011 Selection marker nationallicence Zeocin nationallicence Mammalian cells nationallicence Mutant selection markers nationallicence Error Prone PCR nationallicence Recombinant gene expression nationallicence Van Blokland H. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut Hoeksema F. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut Siep M. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut Otte A. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut Verhees J. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut Cytotechnology Springer Netherlands 63/4(2011-08-01), 371-384 0920-9069 63:4<371 2011 63 10616 https://doi.org/10.1007/s10616-011-9354-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10616-011-9354-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Van Blokland H. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut NATIONALLICENCE 700 1- Hoeksema F. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut NATIONALLICENCE 700 1- Siep M. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut NATIONALLICENCE 700 1- Otte A. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut NATIONALLICENCE 700 1- Verhees J. Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands aut NATIONALLICENCE 773 0- Cytotechnology Springer Netherlands 63/4(2011-08-01), 371-384 0920-9069 63:4<371 2011 63 10616 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer