Protamine enhances the efficiency of liposome-mediated gene transfer in a cultured human hepatoma cell line.
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. Protamine 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 micrograms was the minimal amount of protamine sulfate needed to completely bind 5 micrograms of a plasmid containing a reporter gene, green fluorescent protein (GFP). For transfection assays, protamine (0, 10, 50, 100, and 500 micrograms) was added to a DNA-liposome mixture (5 micrograms DNA and 20 micrograms of a mixed formulation of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate and dioleoylphosphatidylethanolamine) 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, 10 micrograms being most efficient) addition to the liposome-DNA mixture significantly increases the EI, and transfection efficiency of GFP in Huh7 cells.
Ni
Y H
YH
Department of Pediatrics, College of Medicine, National Taiwan University, Taipei, Taiwan.
Hsu
H Y
HY
Chen
P J
PJ
Chang
M H
MH
D000818:Animals; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008081:Liposomes; D008114:Liver Neoplasms, Experimental / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D011479:Protamines; D014162:Transfection; D014407:Tumor Cells, Cultured
D008081:Liposomes; D008164:Luminescent Proteins; D011479:Protamines; D049452:Green Fluorescent Proteins