Joseph C. Wu

Publication Details

  • Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging. FASEB journal Krishnan, M., Park, J. M., Cao, F., Wang, D., Paulmurugan, R., Tseng, J. R., Gonzalgo, M. L., Gambhir, S. S., Wu, J. C. 2006; 20 (1): 106-108

    Abstract:

    Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.

    View details for PubMedID 16246867

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