|Title:||Protamine Enhances the Efficiency of Liposome-Mediated Gene Transfer in a Cultures Human Hepatoma Cell Line||Authors:||NI, YEN-HSUAN
|Keywords:||gene transfection;protamine;liposome;green fluorescent protein||Issue Date:||1999||Journal Volume:||v.98||Start page/Pages:||562-6||Source:||JOURNAL OF THE FORMOSAN MEDICAL ASSOCIATION||Abstract:||
Protamine, used clinically as an antidote for heparin, is a small protein with high arginine content and is potent in folding DNA. Protamine and DNA can form a compact structure, protecting DNA from digestion by intracellular enzymes. Protmine may, therefore, enhance the efficiency of gene transfer. In this study, we tested the ability of protamine to improve liposome-mediated gene transfer efficiency in a human hepatoma cell line. The results of a preliminary gel retardation assay indicated that 10 μg was the minimal amount of protamine sulfate needed to completely bind 5 μg of a plasmid containing a reporter gene, green fluorescent protein (GFP). For transfection assays, protamine (0,10,50, 100, and 500 μg) was added to a DNA-liposome mixture (5μg DNA and 20μg of a mixed formulation of 2, 3-dioleyloxy-N-[2 (sperminecarboxamido) ethyl ]-N, N-dimethyl-l-propanaminium trifluoroacetate and dioleoylphosphatidyletha-nolamine) to transfect cultured Huh7 cells. Transfected cells (those expressing GFP) were counted by using flow cytometry. The expression index (EI) was calculated as the transfection efficiency (% of transfected cells) with protamine divided by the transfection efficiency with DNA and liposome only. Our results show that protamine sulfate ( in a range of 10- 100, 10μg being most efficient) addition to the liposome- DNA mixture significantly increases the EI, and transfection efficiency of GFP in Huh7cells.
|Appears in Collections:||醫學系|
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