Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells.
The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.
Shoji
Jun'ichi
J
Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Tanihara
Yuko
Y
Uchiyama
Tsuneo
T
Kawai
Akihiko
A
D000818:Animals; D006224:Cricetinae; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D008081:Liposomes / Q000008:administration & dosage* / Q000737:chemistry; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014721:Vesicular stomatitis Indiana virus / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000008:administration & dosage / Q000737:chemistry
D008081:Liposomes; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins