In search of expression bottlenecks in recombinant CHO cell lines—a case study (original) (raw)
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Cell Technology for Cell Products, 2007
Recombinant CHO cell lines have integrated the expression vectors in various parts of the genome leading to different levels of gene amplification, productivity and stability of protein expression. Identification of insertion sites where gene amplification is possible and the transcription rate is high may lead to systems of sitedirected integration and will significantly reduce the process for the generation of stably and highly expressing recombinant cell lines. We have investigated a broad range of recombinant cell lines by FISH analysis and Giemsa-Trypsin banding and analysed their integration loci with regard to the extent of methotrexate pressure, transfection methods, promoters and protein productivities. To summarise, we found that the majority of our high producing recombinant CHO cell lines had integrated the expression construct on a larger chromosome of the genome. Furthermore, except from two cell lines, the exogene was integrated at a single site. The dhfr selection marker was colocalised to the target gene.
Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells
Journal of Biotechnology, 2001
Dihydrofolate reductase (DHFR) based amplification of recombinant genes using increasing concentrations of methotrexate (MTX) is a common method to establish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates are isolated, further characterization of their transcriptional pattern is intended to clarify the question what other factors are elevated, as a prerequisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rates, we have investigated alterations in gene expression which occur beside amplification of the recombinant genes. For this purpose, the suppression subtractive hybridization method was used to create a cDNA library enriched for differentially expressed sequences in the recombinant antibody producing CHO cell line versus the original counterpart. Differential expression was confirmed by Northern blotting and Northern ELISA. In addition to the expected recombinant genes, we have identified 5 transcripts which are upregulated in the recombinant cell line. One sequence has not been found in existing data bases, the others revealed to be genes involved in protein synthesis and regulation of transcription. Furthermore, an alternatively spliced, non-functional form of the DHFR mRNA was detected, suggesting a dramatic increase of the selection pressure exerted by MTX.
ACS Synthetic Biology
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Journal of Biotechnology, 2021
Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here, we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell enrichment. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.
Journal of Biotechnology, 2019
Highlights A cumate-inducible expression system is used to generate stable CHO pools expressing high levels of a recombinant protein. Selection in the "on-mode" increases cell mortality compared to selection in the "off-mode" Stable CHO pools generated in the "off-mode" show twofold higher volumetric productivity compared to the pool selected in the "on-mode" The pool selected in the "off-mode" contains a much higher proportion of high-producing cells compared to the pool selected in the "on-mode"
Identifying key signatures of highly productive CHO cells from transcriptome and proteome profiles
Microbial Cell Factories, 2006
One of the key challenges in biotherapeutics production is the selection of a high-producing animal cell line to maximize protein yield in cell culture. Clone selection is often a tedious process, involving rounds of selection and single cell cloning which is costly in both money and time. In an effort to increase the throughput of clone selection, we seek to identify key signatures of a highly productive cell line using an integrated genomic and proteomic platform. In our study, we analysed microarray and proteomics data generated from a characterization of two populations of CHO cells stably expressing high and low levels of green fluorescent protein (GFP). The high producer cells (HP) make 6x more GFP than the low producer cells (LP) as determined by ELISA. Comparison of transcript levels between HP and LP in the mid-exponential phase was performed using a proprietary 15k CHO cDNA microarray chip, of which 7559 genes are unique [1], while proteomic analysis on samples in the mid-exponential and stationary phases was performed using iTRAQ quantitative protein profiling technique . Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization.
BMC biotechnology, 2015
Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of...