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Publication Details
Year
:
2014
Journal
:
Kamal Shaik Fakiruddin, Puteri Baharuddin, Moon Nian Lim, Noor Atiqah Fakharuzi, Nurul Ain Nasim and Zubaidah Zakaria (2014). Efficient TRAIL gene delivery using nucleofection based method in human adiposed derived mesenchymak stromal cells. Cell Biology 2(1): 28-33
Abstract
:
Being able to perform genetic manipulation of human adipose-derived mesenchymal stromal cells (ADMSCs) will harness the benefits of these cells beyond degenerative diseases. Most primary cells show resistance to genetic alteration with viral transduction remains to be the most effective tool for gene delivery. However, the use of viral vectors has several disadvantages mainly involving safety risk. Here, we report optimization using safe and yet efficient nucleofection based transfection of DNA plasmid encoded for TNF-related apoptosis inducing ligand (TRAIL) into ADMSCs. Initial characterization of ADMSCs was performed based on cells morphological evaluation and surface protein expression. Nucleofection revealed 10% higher transfection efficiency compared to lipofection (Fugene 6 and Turbofect) with optimal cells viability (~87%). Subsequent nucleofection analysis showed the increased plasmid concentration of 10µg resulted in significantly higher reporter expression with 35% efficiency and 43% yield. Transgene expression was stable at day 9 with 74% cells remained to be GFP+, but was reduced to baseline at day 15. In this report, we have showed that the nucleofection technique is efficient to deliver exogenous gene in ADMSCs compared to common lipofection methods. We also noticed that increased plasmid concentration enhanced nucleofection efficiency and yield in ADMSC. Furthermore, exogenous expression of the gene was transient with no evidence of stable genomic integration, thus we concluded that the nucleofection technique is an efficient and yet safe nonviral transfection technique in ADMSCs.
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