title: Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells.
abstract: BACKGROUND
  Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing.
  
  
  RESULTS
  While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome. A greater proportion of cells in clones generated using the CpGfree promoter were still expressing detectable levels of EGFP after 8 weeks but the relative expression levels measured at week 8 to those measured at week 0 did not improve compared to clones generated using the CpGrich promoter. Chromatin immunoprecipitation assays indicated that the repression of the CpGfree promoter was likely linked to histone deacetylation and methylation. Use of histone deacetylase inhibitors also managed to recover some of the lost expression.
  
  
  CONCLUSIONS
  Using a promoter without CpG dinucleotides could mitigate the early gene silencing but did not improve longer-term expression stability as silencing due to histone modifications could still take place. The results presented here would aid in promoter selection and design for improved protein production in CHO and other mammalian cells.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Steven C L
authors.initials: SC
authors.lastname: Ho
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Esther Y C
authors.initials: EY
authors.lastname: Koh
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Benjamin P C
authors.initials: BP
authors.lastname: Soo
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.lastname: Mariati
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.|Department of Microbiology, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore, 117597, Singapore.
authors.forename: Sheng-Hao
authors.initials: SH
authors.lastname: Chao
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore. yang_yuansheng@bti.a-star.edu.sg.
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000818:Animals; D016466:CHO Cells; D018899:CpG Islands / Q000235:genetics*; D003412:Cricetulus; D004355:Drug Stability; D024861:Genetic Enhancement / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins
keywords: CHO cells; DNA methylation; Gene silencing; Histone modifications; Recombinant protein expression