Recent advances in genome editing technology have brought genome engineering in mammalian cells closer to reality. This has spurred much new efforts in the exploration of new ways of generating production cell lines for therapeutic proteins. Increasingly, cell biotechnologists are exploring targeted integration of the product transgene directly into a specific locus of the genome. It has long been the doctrine of cell line development that the transcript level of the transgene is pivotal to high productivity. The use of strong promoters to drive the expression of the transgene and gene amplification to increase the copy number has long been employed to increase the transcription level. However, our knowledge of transcriptional activity and regions of a genome still relatively sparse and defining such regions had been laborious and costly until high throughput sequencing cost has reduced substantially recently. In this study, we investigated genome-wide distribution of transgene expression in Chinese hamster ovary (CHO) cells. We generated libraries of CHO cells with a single integration site of a vector that expresses green fluorescent protein (GFP) as the reporter of transcript level. After sorting according to GFP fluorescence intensity, cells with high transcript levels were isolated and the segments of the genomic DNA containing the integration site of transgene were PCR enriched and subjected to sequencing. Our interest on the positional effect of the transgene integration loci is not limited to the transcriptional activity but also the propensity that the transcriptional activity is suppressed or silenced due to epigenetic events. Such transcriptional suppression will transpire in the instability of the cell line. The reporter is again a valuable tool in the investigation of transgene expression instability. The results of this genome wide survey will provide a better understanding of the influence of genome regions or even genome context on transgene expression and facilitate the application of genome engineering technology in cell line development.

Genome-wide analysis of transgene expression in Chinese hamster ovary cells

249th National Meeting (2015)

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