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bioRxiv · 10.64898/2026.07.22.739919

Epigenetic Regulation of Stable SARS-CoV-2 RBD-sfGFP Expression in Primary Human Splenic Fibroblasts

Maan, K. S.·Baloch, Z. A.·Bhullar, S. S.·Vashishat, I.·Assogba, B. D.
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Abstract

BackgroundRecombinant expression of the SARS-CoV-2 receptor-binding domain (RBD) is essential for vaccine development, serological diagnostics, and mechanistic studies. Primary human fibroblasts offer physiologically relevant protein folding and post-translational modification, yet their short lifespan limits scalable production. We used an immortalized human splenic fibroblast cell line to stably express RBD-sfGFP for longitudinal characterization and downstream studies. MethodsImmortalized human primary splenic fibroblasts were transfected by electroporation with a plasmid encoding SARS-CoV-2 RBD fused to superfolder GFP (sfGFP), with a neomycin resistance cassette (neoR) for G418 selection. Four independent G418-resistant cultures (n=4), designated HPSF-IM-RBD-BHSKPU T1-T4, were established from distinct selection flasks. Based on previous screenings, two cultures (T1, T3) were monitored for 98 days (14 passages, P1-P14); two cultures (T2, T4) were monitored for 42 days (6 passages, P1-P6). RBD-sfGFP expression was assessed by fluorescence microscopy at 7-day intervals. For each timepoint, 2 fields were imaged and analyzed for relative fluorescence intensity (normalized to global maximum = 100%) and mean fluorescence intensity (MFI, normalized to global maximum = 100%). Coefficient of variation (CV), linear regression, and Pearson correlation were calculated. ResultsAll four cultures exhibited robust GFP fluorescence, confirming stable transgene retention. Expression ranking: T1 (93.1% +/- 3.6%) > T3 (89.2% +/- 3.4%) > T2 (84.2% +/- 3.2%) > T4 (79.7% +/- 3.9%). Long-term cultures T1 and T3 retained [~]100% of Day 7 signal at Day 98 (T1: 100.7%; T3: 100.0%). Expression exhibited passage-dependent oscillation rather than progressive silencing. CV increased over time in T1 (1.5% -> 8.5%), indicating growing inter-cellular heterogeneity. A strong positive correlation between fluorescence and MFI (Pearson r = 0.823, p = 7.44 x 10-11) suggested coherent population-level regulation. ConclusionsHPSF-IM-RBD-BHSKPU cells stably retain RBD-sfGFP expression for over 3 months, validating their utility as a recombinant protein production platform. However, oscillatory dynamics and increasing heterogeneity are consistent with position-effect variegation at distinct integration loci. Consequently, early passages (P1-P4) are optimal for applications requiring maximal uniformity. Ultimately, these cells provide a practical tool for RBD production and a valuable model for studying epigenetic regulation of transgene expression in human primary fibroblast backgrounds.

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Maan, K. S., Baloch, Z. A., Bhullar, S. S., Vashishat, I., Assogba, B. D.. 2026-07-23. Epigenetic Regulation of Stable SARS-CoV-2 RBD-sfGFP Expression in Primary Human Splenic Fibroblasts. https://doi.org/10.64898/2026.07.22.739919

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