We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a plasmid carrying a loxP-green fluorescent protein (GFP) fusion gene and a DHFR gene, we screened colonies by fluorescent intensity. We selected 16 clones that expressed high levels of GFP and carried one copy of the plasmid in their chromosomes and treated them with methotrexate (MTX) to examine their ability for DHFR-mediated gene amplification. Two clones, MK1 and MK2, showed increased GFP expression upon gene amplification. In those clones, the loxP-GFP gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a loxP-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal loxP by Cre recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. In human monoclonal antibody production, after gene-targeting of loxP in MK2 and gene amplification with MTX, the MTX-resistant colonies showed high levels of antibody production. The most productive clone was able to produce 160 mg/l in 7 days in a low-protein medium in a spinner-flask.
In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.
Cyclophilin is a ubiquitous peptidyl prolyl cis/trans isomerase that plays critical roles in many biological processes. A number of cyclophilin inhibitors have been designed based on the structure of the immunosuppressant cyclosporin A. To discover inhibitors that have other structures, the authors established the high-throughput screening (HTS) method using FDSS6000 real-time fluorescence detector. The inhibitors identified with this HTS showed significant correlation with direct interaction as measured by surface plasmon resonance. This high-throughput assay system is a powerful tool for the discovery of peptidylprolyl isomerase inhibitors. (Journal of Biomolecular Screening 2009:419-424)
ABSTRACT:In order to obtain a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, five kinds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cells were compared with each other, and the distribution of the location of amplified genes was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell line obtained under the selection condition in which the stepwise increase in the MTX concentration was most gradual reached the highest levels and under this condition about 80% of the amplified genes were observed near the telomeric site. During long-term cultivation without MTX, the percentage of amplified genes near the telomeric site hardly changed, but that of amplified genes at other sites decreased. The specific production rate gradually decreased during cultivation without MTX. To clarify the relationship between the specific production rate and the location of amplified genes, a cloned cell line, DR1000L4N, was obtained. This cell line showed higher productivity, and the amplified genes were all situated near the telomeric site.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.