The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus) liposomes with gangliosides

Abstract

Macromolecules such as DNA and RNA can be entrapped within liposomes associated with gangliosides by reverse-phase evaporation. When these liposomes are incubated with HVJ 2 2 Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DNase I, deoxyribonuclease I; RNase, ribonuclease; PS, phosphatidylserine; PC, phosphatidylcholine; Chol, cholesterol; PE, phosphatidylethanolamine; GS, gangliosides; REV, reverse-phase evaporation; HVJ, hemagglutinating virus of Japan (Sendai virus); HVJ-GSL, HVJ liposomes associated with gangliosides; ETC, Ehrlich ascites tumor cells; Ig, immunoglobulin; MEM, minimum essential medium; TK, thymidine kinase; BSS, buffered salt saline; PBS, phosphate-buffered saline. (Sendai virus), they deliver their contents into cultured cells efficiently. More than 95% cells of a Ltk − cell line (thymidine kinase-deficient cells) transiently expressed thymidine kinase activity by thymidine kinase gene transfer using HVJ liposomes with gangliosides. Stable transformants could be obtained efficiently from various cell lines by use of HVJ liposomes containing the neo r gene. The neo + transformants were obtained at frequencies of about 0.2–1.0, 0.06–0.25, and 0.06–0.1% in monolayers of L, CHO-K1, and HeLa-S3 cells, respectively. Moreover, in Ehrlich ascites tumor cells which grow in suspension, the frequency was more than 0.01%. On introduction of plasmid pTK4 into Ltk − cells, about 0.5–1.0% TK + transformants were obtained. Cosmid DNA containing the neo r gene (about 45 kbp) was also introduced into L cells by this method and neo + transformants were obtained at a frequency of 0.1%. When rat liver mRNA was introduced into L cells by HVJ liposomes with gangliosides, immunoprecipitation studies showed that the L cells secreted rat albumin and some other proteins into the cultured medium. Moreover, using erythrocyte membrane vesicles containing IgM that had been incubated with HVJ empty liposomes with gangliosides, the IgM could be introduced into all the L cells.