Salman M, Tarshis M, Rottem S
Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
J Bacteriol. 1992 Jul;174(13):4410-5. doi: 10.1128/jb.174.13.4410-4415.1992.
We have developed and characterized a system for the transfer of plasmids encapsulated in large unilamellar vesicles (LUV) into Spiroplasma floricola BNR1 cells. The approach is based on the ability of S. floricola-derived LUV to fuse with S. floricola cells. The fusion was continuously monitored by an assay for lipid mixing based on the dequenching of the fluorescent probe octadecylrhodamine B (R18) that was incorporated into LUV at self-quenching concentrations. The fusion was also evaluated by fluorescence-activated cell sorter measurements and by sucrose density gradient analysis. LUV-cell fusion occurred only in the presence of low concentrations (5%) of polyethylene glycol (polyethylene glycol 8000) and depended on temperature, the LUV/cell ratio, and divalent cations in the incubation medium. Throughout the fusion process, spiroplasma cells remained intact and viable. Under optimal fusion conditions, the plasmid pACYC, encapsulated in LUV by reversed-phase evaporation, was transferred into live S. floricola cells and expressed chloramphenicol acetyltransferase activity. The expression was transient with maximal chloramphenicol acetyltransferase activity observed after 6 h of incubation of the transfected cells.
我们已经开发并表征了一种将包裹在大单层囊泡（LUV）中的质粒转移到弗罗里达螺原体BNR1细胞中的系统。该方法基于弗罗里达螺原体来源的LUV与弗罗里达螺原体细胞融合的能力。通过基于掺入LUV中处于自猝灭浓度的荧光探针十八烷基罗丹明B（R18）的去猝灭的脂质混合测定法持续监测融合情况。还通过荧光激活细胞分选仪测量和蔗糖密度梯度分析来评估融合情况。LUV-细胞融合仅在低浓度（5%）的聚乙二醇（聚乙二醇8000）存在下发生，并且取决于温度、LUV/细胞比例以及孵育培养基中的二价阳离子。在整个融合过程中，螺原体细胞保持完整且存活。在最佳融合条件下，通过反相蒸发包裹在LUV中的质粒pACYC被转移到活的弗罗里达螺原体细胞中并表达氯霉素乙酰转移酶活性。该表达是短暂的，在转染细胞孵育6小时后观察到最大的氯霉素乙酰转移酶活性。
