ObjectivesTo identify genes that affected protein expression in Chinese hamster ovary (CHO) cells was significant, and we identified the changes in the transcriptome and the functional gene sets that would contribute to increase expression of recombinant protein.ResultsHere two sub-clones from a methotrexate-treated parental recombinant CHO cell line were selected. The two sub-clones, with different expression levels (qp were 42.8pg/cell/day and 14.0pg/cell/day), were analyzed through RNA-seq. More than 600 genes were identified as differently expressed, and we found that the differentially expressed genes were involved in processes such as RNA processing, transcription, protein catabolism, and protein transport. Among these, we cloned genes encoding proteins that were involved in transcription and protein transport to investigate their effect on protein production.ConclusionsWe found that some genes involved in transcription and protein transport would improve recombinant protein production in CHO cells.