Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification

Abstract

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into>1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

“…Low recombinant protein expression levels and transgene silencing are common issues in current recombinant protein production and can be caused by positional effects related to neighbouring chromatin . To overcome these issues, gene regulatory elements, such as insulators, ubiquitous chromatin opening elements, expression augmenting sequence elements, stabilizing and anti‐repressor elements, and MARs , are used to increase recombinant protein production.…”

Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference

“…Future studies will be directed toward employing multiple gRNA targeting different regions of dhfr promoter and open reading frame to achieve tighter knockdown of dhfr transcription, which may allow us to generate producer clones with better protein yield after MTX selection. In addition, several other different approaches have been developed to enhance recombinant protein production in CHO cells, such as using stronger promoters to drive the exogenous gene, overexpressing cell cycle regulating protein, inserting matrix attachment regions , and cointegrating Ubiquitin Chromatin Opening Elements to modify the chromatin structures. Conversely, other vectors such as transposon enables more efficient gene integration and may increase the initial copy number of dhfr and gene of interest.…”

A short synthetic chimeric sequence harboring matrix attachment region/PSAR2 increases transgene expression in Chinese hamster ovary cells

“…15) Several chromatin functionmodifying elements, including the matrix attachment region (S/MAR), internal ribosome entry site (IRES), and ubiquitous chromatin opening element, can also affect the chromatin environment and enhance transgene expression. [16][17][18][19] MARs were first identified in DNA fragments that were retained in nuclear scaffold/matrix preparations. 20) MARs are typically AT-rich (>60%) and have several short consensus sequences, such as an A-box (AATAAAYAAA), T-box (TTWTWTTWTT), DNAunwinding sequence (AATATATTT or AATATT), and topoisomerase II-binding site (GTNWAYATT-NATNNR).…”