Promotör Bağımsız CpG Zengini A2UCOE Alt Bölgelerinin Transgen Ekspresyonu Üzerine Etkisi

Abstract

Amaç: Bu çalışmanın amacı, HNRPA2B1 ve CBX3 promotörleri hariç olmak üzere, A2UCOE elemanındaki CpG zengin alt bölgelerin lentiviral sistemlerde uzun süreli transgen ekspresyonunu sürdürme yeteneğini değerlendirmektir. Bu bölgeler spleen focus-forming virüs (SFFV) promotörünün önüne klonlanmış ve P19 ile F9 hücrelerinde test edilmiştir. Gereç ve Yöntemler: Bu çalışmada, promotör içermeyen üç A2UCOE alt bölgesinin ubiquitous chromatin opening element (UCOE) aktivitesi değerlendirildi. P19 ve F9 fare embriyonal karsinom hücreleri kullanılarak, farklılaşmamış ve farklılaşmış durumlarda enhanced green fluorescent protein (eGFP) ekspresyon stabilitesi ve dayanıklılığı incelendi. CpG zengin, promotörsüz fragmanlar içeren lentiviral vektörler (LV’ler), 455UCOE (OFA1), 527UCOE (OFA2) ve 945UCOE (OFA3) hücrelere aktarıldı ve akım sitometrisiyle sürekli transkripsiyonel aktivite analiz edildi. Bulgular: Bazı vektörler eGFP ekspresyonunu sürdürürken bazıları ise hem farklılaşmamış hem de farklılaşmış durumlarda hızlı bir azalma gösterdi. Özellikle OFA1, OFA2 ve OFA3 vektörleri, SEW vektörüne benzer eğilimler göstererek zamanla eGFP ekspresyonunda belirgin azalmalar sergiledi. CBX3 kökenli fragmentlerin hiçbiri stabil ekspresyon sağlamadı, bu da UCOE aktivitesinin olmadığını düşündürdü. Ekspresyon azalma hızları, SFFV promotörlü SEW vektörü ile OFA1, OFA2 ve OFA3 içeren LV’ler arasında benzerdi. Sonuç: CpG zengin, promotör içermeyen fragmanlar fare embriyonal karsinom hücrelerinde stabil transgen ekspresyonunu destekleyememekte, bu da UCOE fonksiyonelliğinin olmadığını göstermektedir. Bulgular, gen terapisi için açık kromatinin korunmasında promotör ve düzenleyici elemanların önemini vurgulamaktadır. Gelecek çalışmalar, transgen stabilitesini artırmak için alternatif CpG zengin bölgeler veya minimal promotörleri araştırmalıdır.

Keywords

• Promotor aktivitesi
• UCOE Fonksiyonu
• trasnkripsiyonel susturma
• SFFV promotörü
• Gen ekspresyonu stabilitesi

Impact of Promoter-Independent CpG-Rich A2UCOE Subregions on Transgene Expression

Abstract

Aim: This study aimed to evaluate the ability of CpG-rich subregions within the A2UCOE element, excluding the HNRPA2B1 and CBX3 promoters, to sustain long-term transgene expression in lentiviral systems. These regions are cloned upstream of the spleen focus-forming virus (SFFV) promoter and tested in P19 and F9 cells. Material and Methods: The study assessed the ubiquitous chromatin opening element (UCOE) activity of three A2UCOE subregions that lack promoters. Using P19 and F9 murine embryonal carcinoma cells, enhanced green fluorescent protein (eGFP) expression stability and durability were examined in undifferentiated and differentiated states. Lentiviral vectors (LVs) containing CpG-rich, promoterless fragments, 455UCOE (OFA1), 527UCOE (OFA2), and 945UCOE (OFA3), were transduced into the cells, and sustained transcriptional activity was analyzed via flow cytometry. Results: Some vectors sustained eGFP expression, while others exhibited a rapid decline in both undifferentiated and differentiated states. Specifically, the OFA1, OFA2, and OFA3 vectors showed trends similar to the SEW vector, with significant reductions in eGFP expression over time. None of the CBX3-derived fragments provided stable expression, suggesting no UCOE activity. The rates of expression decline were similar between the SEW vector with the SFFV promoter and the LVs containing OFA1, OFA2, and OFA3. Conclusion: CpG-rich, promoterless fragments failed to support stable transgene expression in murine embryonal carcinoma cells, indicating a lack of UCOE functionality. These findings highlight the role of promoter and regulatory elements in maintaining open chromatin for gene therapy. Future studies should investigate other CpG-rich regions or minimal promoters to improve transgene stability.

Keywords

• Promoter activity
• UCOE function
• Transcriptional silencing
• SFFV promoter
• Gene expression stability

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