Then, stable cell populations exhibiting stable transgene integration were cultured in CD CHO medium supplemented with 8 mM l‐glutamine in 125‐ml Corning shake flasks with 30 ml medium with the presence of 500 μg/ml G418 for 30 days. At 48 hrs after transfection, stable transfected cells were selected by 800 μg/ml geneticin selection for 2 weeks until positive colonies appeared. ( A) Relative eGFP mRNA levels in stable transfected cells. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.Įffect of different introns on the production of eGFP in stable transfected CHO‐S cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.Ĭhinese hamster ovary (CHO) gene expression gene regulation intron. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. And gene copy number was also detected by qPCR, respectively. qRT-PCR and flow cytometry were used to explore eGFP expression levels. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. However, the product yield and transgene instability need to be further increased and solved.
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