earticle

영어

Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. To increase productivity of rCHO cells, gene amplification system, adding stepwise increment of methotrexate, is generally utilized. The clone did not show im proved productivity proportional to increased MTX concentration, which can be called as rate-limiting step. We speculated that promoter methylation may be one reason, which limits full production capacity. Generally, in mammalian cells, cytomegalovirus (CMV) promoter is used for transcription of gene of interest. 36 CpG sites were identified within CMV promoter, which are candidates for de novo methylation. Here, we hypothesize that methylationat CMV promoter might lead to reduced gene transcription. It is well known that methylation of DNA inhibits gene expression. The results indicated that specific productivity decreased at 320nM amplified clone compared to 80 nM amplified clone. Significant methylation difference at CMV promoter was identified between parental cloneand MTX amplified clones. For future studies, we will focus on correlating promoter methylation with transcription level. To support the hypothesis, we will analyze gene copy number through real-time PCR. Taken together, we provide first hints that amplified gene copy number could be silenced by promoter methylation, which can be proved by reduced mRNA transcription.