A novel, high stringency selection system allows screening of few clones for high protein expression
| Autor: | John Antonius Verhees, Tjalling Siersma, S.Y. Man, H. J. M. Van Blokland, Ted H.J. Kwaks, C. van Mer, S. Botschuijver, Richard George Antonius Bernardus Sewalt, Vincent Klaren, N.C. Teunissen, Arie P. Otte, J.W.M. Korse |
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| Přispěvatelé: | Epigenetic Regulation of Gene Expression (inactive) (SILS, FNWI) |
| Rok vydání: | 2007 |
| Předmět: |
Cloning
Genetics Low protein High protein Gene Dosage Bioengineering CHO Cells General Medicine Biology Transfection Applied Microbiology and Biotechnology Cell Line Cricetulus Eukaryotic translation Gene Expression Regulation Cell culture Cricetinae Animals Cloning Molecular Gene Selection (genetic algorithm) Biotechnology Selection system |
| Zdroj: | Journal of Biotechnology, 128(2), 237-245. Elsevier |
| ISSN: | 0168-1656 |
| Popis: | To obtain highly productive mammalian cell lines, often large numbers of clones need to be screened. This is largely due to low selection stringencies, creating many, but low protein producing clones. To remedy this problem, a novel, very stringent selection system was designed, to create few, but high protein producing clones. In essence, a selection marker with a startcodon that confers attenuated translation initiation frequency was placed upstream of the gene of interest with a startcodon that confers optimal translation initiation. From the transcribed bicistronic mRNA, the selection marker is translated at a low frequency, and the protein of interest at a high frequency. This selection system is so stringent that clones form only rarely. However, application of anti-repressor elements, which increase promoter activity, did induce the formation of clones that expressed proteins at high levels. When combined with anti-repressor elements, this novel selection system can be a valuable tool to rapidly create few, but highly productive mammalian cell lines. |
| Databáze: | OpenAIRE |
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