Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity.
We developed an expression system that aimed to increase the proportion of high producers in a transfected cell population in order to reduce the effort in clone screening. The principle is based on the impairment of the selection marker. Twelve single-point mutations in more or less conserved domains of the resistance marker gene neomycin-phosphotransferase (NPT) resulted in different degrees of reduced enzyme activity, depending on the amino acid conservation and the kind of amino acid exchange. In all transfected, mutant-NPT bearing CHO-DG44 cell pools surviving the selection with G418, the ratio of high-producing cells to total cell number was higher than in pools selected with wildtype-NPT. Furthermore, these pools showed, in comparison to wildtype-NPT selected pools, not only higher NPT-RNA levels but also increased specific productivities and higher titers of a coexpressed biopharmaceutically relevant product. Elevated productivity could be ascribed to higher gene copy numbers, integration into chromatin regions with higher transcriptional activity, or a combination of both effects. Thus, the use of NPT-mutants as selection markers is suitable for the enrichment of high producers in a transfected CHO-DG44 cell population, since cell survival is achieved only if the enzymatic impairment of the cointegrated resistance marker is compensated by a higher expression level.
Sautter
Kerstin
K
Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, D-88397 Biberach/Riss, Germany.
Enenkel
Barbara
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D018932:Chemokine CCL2 / Q000378:metabolism; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression / Q000187:drug effects; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012641:Selection, Genetic; D014162:Transfection
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D005819:Genetic Markers; D005839:Gentamicins; D011994:Recombinant Proteins; C010680:antibiotic G 418; D019868:Kanamycin Kinase