Ali Osman Arslan, Ömer Faruk Anakök
Northwestern Medical Journal - 2025;5(4):222-233
Aim: The A2UCOE, a well-characterized chromatin-modifying element originating from a constitutively expressed human gene locus, promotes consistent and durable gene activity, even when foreign DNA sequences are inserted into densely packed heterochromatin regions. The aim of this study is to examine the Daedalus fragment in A2UCOE to identify key sub-regions responsible for its prominent chromatin opening activity. This would enable the discovery of a minimal yet fully functional element, allowing for more efficient utilization of vector capacity to accommodate therapeutic transgenes when incorporated into lentiviral vectors (LVs). Method: To assess the chromatin-opening activity of the Daedalus sub-region within the A2UCOE, lentiviral vectors carrying eGFP reporter constructs were generated and transduced into murine embryonal carcinoma cell lines (P19 and F9). Transgene expression stability was assessed across both pluripotent cells and those differentiated with retinoic acid. HEK293T cells were used for lentiviral packaging. Plasmid propagation was performed in E. coli DH5alpha, and standard molecular cloning techniques were applied. Flow cytometry was used to measure eGFP expression levels, and significance was assessed via Student's t-test (p < 0.05). Results: Flow cytometry and RT-qPCR analyses revealed that the positive control vector (1.5A2UCOE-SEW) maintained stable eGFP expression in both differentiated states of P19 and F9 cells, whereas the UCOE vectors named Daedalus-F and Daedalus-R were unable to prevent transgene silencing. In undifferentiated cultures, eGFP expression from these vectors decreased by 40-50% within two weeks, with a similar decline observed in differentiated cells. The average vector copy numbers remained stable, indicating transcriptional silencing rather than vector loss. Conclusion: The Daedalus sub-region of A2UCOE alone could not prevent transgene silencing in pluripotent or differentiated cells, indicating additional A2UCOE elements are needed for stable long-term expression.
