Yan-Long Jia1,2, Xiao Guo1,2, Xi-Cheng Wang1,2, Tian-Yun Wang3,4. 1. School of Pharmacy, Xinxiang Medical University, Xinxiang, 453003, Henan, China. 2. International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, 453003, Henan, China. 3. International Joint Research Laboratory for Recombiant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, Xinxiang, 453003, Henan, China. wtianyuncn@126.com. 4. School of Basic Medicine, Xinxiang Medical University, Xinxiang, 453003, Henan, China. wtianyuncn@126.com.
Abstract
OBJECTIVES: To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. RESULTS: Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. CONCLUSIONS: The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.
OBJECTIVES: To investigate the effect of full-length fragment of DNA topoisomerase I gene (TOP1) matrix attachment regions (MARs) originating from the human genome on transgene expression in Chinese hamster ovary (CHO) cells and explore the underlying mechanisms. RESULTS: Results showed that TOP1 MAR cannot only enhance the transient and stable transgenic expression of enhanced green fluorescence protein (EGFP) but also increase long-term stability and ratio of positive colonies in transfected CHO cells with TOP1 MAR at the 5' or 3' ends of the EGFP expression cassette. Interestingly, the CHO cells were transfected with the 5',3' TOP1 MAR-containing vector featured the highest transient and stable expression, whereas those with the 3' TOP1 MAR-containing vector exhibited the most effective stability and ratio of positive colonies. We also observed that transgene copy numbers and mRNA of egfp gene were correlated with the expression levels of EGFP protein in polyclonal CHO cells. However, the heterogeneity of expression in monoclonal CHO cells was unaffected by transgene copy number. CONCLUSIONS: The findings may aid in the potential application of TOP1 MAR in expression enhancement of recombinant proteins in mammalian cells.
Entities:
Keywords:
Chinese hamster ovary cells; Human DNA topoisomerase I gene (TOP1); Matrix attachment region (MAR); Transgene expression