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Stable cell line generation requires the gene of interest to become stably integrated into the host cells genome. The generation of stable cell lines by random integration produces large variation in clonal expression and stability that is due, in part, to the nature of the chromatin it has integrated into.\ud \ud Elements have been identified that can modify the chromatin structure and have been shown to protect transgcne expression from the effects of the neighbouring chromatin environment. The aim of the study was to investigate whether the identified chromatin structure modifying elements (UCOE, MAR, STAR and HS4) could increase the level and stability of antibody expression in stable cell lines in the mammalian expression systems used at UCB.\ud \ud A series of expression constructs were generated to assess the effect of the chromatin structure modifying elements on antibody expression in stable cell lines. Initially seven different vectors for each clement that possessed different combinations of the individual elements were assessed as pooled and clonal stable cell populations which indicated that the elements increased antibody expression by varying amounts and optimal vector configurations were different for the elements.\ud \ud The optimised vectors were subsequently used in a comparative side-by-side study with expression levels from pooled and stable cell lines being analysed. A subset of clones harbouring each of the five test constructs (Ab535 control, 1.5kb A2UCOE, MAR X_S29, STAR 7 and cHS4 tandem) derived from the pooled stable cell lines were genetically characterised to determine copy number and clonal diversity. Clones were cultured long-term in the absence of selection pressure which resulted in a decrease in expression being observed in all of the clones. The decline in antibody expression was accompanied by a decrease in mRNA levels which was not caused by a loss of either HC or LC gene copies from the genome. Analysis of DNA methylation patterns across the hCMV-MIE promoter regions demonstrated that this epigenetic regulatory mechanism was involved in the silencing of a number of clones which exhibited a decreasein productivity. |
