Use of liposome to transfer plasmid DNA into yeast cells

Abstract

Utilization of liposome as a carrier of transporting genetic materials having relatively large molecular weight such as plasmids into yeast cells was tried in this investigation. Encapsulation of plasmids into liposomes may protect the DNA molecules from degredation. Membrane-membrane fusion may also facilitate transfer of plasmids when the liposomes are contact with yeast protoplasts. Two types of yeast plasmids, YEp13 and pMA56, were entrapped separately into large unilamellar vesicle (LUV) or reversephase evaporation vesicle (REV) liposomes by the methods of ether injection or reversephase evaporation, respectively. Electron microscopic observations proved that the two types of liposomes prepared, LUV and REV, to be relatively homogeneous in size and structure having average diameter of 0.25 $\mu$m. The encapsulation of plasmids in the liposomes was evidenced by Sepharose CL-4B column chromatography using $^3H$-labeled plasmids. Encapsulation efficiencies of REV and LUV were about 50\% and 6\%, respectively. The plasmids entrapped in the liposomes could retain their original state of covalently closed circular form. And the original transformation activities of the entrapped plasmids were also found to be unaltered. These results were based on the experiments, such reisolation of plasmids from the liposomes, electrophoresis, and transformation. The cells of Saccharomyces cerevisiae were converted to protoplasts by treating with Zymolyase 60,000. The enzyme concentration of 0.025 mg/ml was found to be suitable for yeast cells of $5\times10^8$ per ml. The treatment was carried out under hypertonic conditions by adding 1.0M of sorbitol as osmotic stabilizer to protect protoplasts against osmolysis. It was found that the hypertonic conditions provided to protect naked protoplasts reduced the rate of regeneration of the same protoplast substantively. The inhibitory effect could be avoided partially by growing the yeast cells under the same hypertonic conditions a...