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Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts

Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-ken 305, Japan

Reverse-phase evaporation lipid vesicles (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 1:3 molar ratio, encapsulated approximately 30% of exogenously supplied recombinant DNA vector, pBR322. The DNA sequestered in REV liposomes was highly tolerant to DNase.

A two-step procedure was developed, which involves encapsulation of DNA with liposomes using one-tenth phosphate-buffered saline-0.5 molar mannitol, followed by incubation of liposome-DNA with protoplasts in phosphate buffer-0.5 molar mannitol.

About 11% of liposome-encapsulated DNA was transferred into protoplasts, whereas 6% uptake was observed in the control. Although some degradation of incorporated DNA occurred inside protoplasts, 50% of the total radioactivity resolved by 0.8% agarose gel was associated with pBR322 forms in 5-hour incubation. After 20-hour incubation, open circular DNA disappeared completely and maintenance of covalently closed circular DNA was confirmed.