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Abstract

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.

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

“…All three cell lines showed different levels of productivity, gene copy number and mRNA content. Previous studies have also reported that despite being derived from a single clone, cell lines can become quite heterogeneous following MTX amplification [11,41,51]. All non-UCOE cell lines had reduced productivity over longterm culture in the absence of MTX, with a *90 % decrease in productivity, which contrasted with a *50 % loss of gene copy that was observed in the absence of MTX.…”

Construction of a gene knockout CHO cell line using a simple gene targeting method

“…To investigate the efficiency of CRISPR-Cas9 gene targeting in CHO cells, the frequency of gene integration into chromosome O was analyzed. In a previous study, chromosome O was identified as a suitable target site for stable and highly efficient exogenous gene expression [ 3 ]. In Figure 1, red signals indicate the BAC clone Cg0031N14 hybridized to chromosome O.…”

FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

“…Early studies [ 14 ] have shown that telomere-type clones, in which the exogenous gene is located near the telomeric region, were found to have a specific hGM-CSF production rate, approximately 6 times higher than the rate of other type of clones. Our study also provided evidence that the telomeric region at chromosome 1 appears to be a desired site for exogenous gene integration.…”

“…Analysis of a broad range of recombinant cell lines and their integration loci found that the majority of highly producing cell lines have an insertion locus on large chromosomes [ 4 , 6 ]. The fragile site and the telomeric region have been proposed to be the preferred integration site of the stable transfectant [ 13 , 14 ]. However, others have suggested that gene integration at known fragile sites can lead to genomic instability, thus affecting the expression of targeted genes [ 15 ].…”