Selection of Fused Cells from Yeast Protoplasts by Flom Cytometry with Dual Fluorescence Labelling

对酵母融合原生质体进行了流式细胞术分析，为其在酵母育种中的应用奠定了基础。在酵母菌中，葡萄糖淀粉酶和α-淀粉酶分别为糖酶和α-淀粉酶。FACStar(Becton Dickinson免疫细胞测定仪)配备细胞分选仪，首先用荧光分光光度计、激光共聚焦显微镜和流式细胞仪对荧光染料进行筛选。异硫氰酸荧光素异构体I和罗丹明6G分别是标记细胞膜和线粒体的最佳异构体。其次，选择具有双荧光标记的融合细胞，在配有显微操作仪的荧光显微镜下收集细胞。第三，将流式细胞术引入上述双荧光标记细胞分选中。对流式细胞仪和细胞分选仪的操作条件进行了研究。分析数百万个融合的酵母原生质体需要很短的时间；例如，分析数百万个细胞大约需要1个小时，分选亚数百万个细胞获得数千个融合原生质体需要1个小时。大约1%的种群被分类，其中1%被再生。通过提取细胞染色体的脉冲场琼脂糖凝胶电泳法和用碘化丙啶标记的流式细胞仪测定细胞DNA含量来确定细胞融合。

Flow cytometry of fused protoplasts of yeasts was investigated for the use in breeding yeasts. The model was glucoamylase and alpha-amylase in yeasts Saccharomyces diastaticus and Saccharomycopsis fibuligera, respectively. Flow cytometer used was FACStar (Becton Dickinson Immunocytometry Systems Inc.), which is equipped with cell sorter.First, fluorescent dyes were screened with use of a fluorescence spectrophotometer, a microcomputer-aided confocal LASER microscope and the flow cytometer. Fluorescein isothiocyanate isomer I and rhodamine 6G were selected, which were the best for labelling cell membrane and mitochondrion, respectivy. The best conditions of labelling were established, and used afterwards.Second, fused cells which had dual fluorescent labels were selected and collected under a fluorescence microscope equipped with micromanipulater. It took a long time to obtain a small number of fused cells.Third, flow cytometry was introduced to the above-mentioned cell sorting with dual fluorescent labelling. Conditions of operating the flow cytometer and cell sorter were studied. It took a short time to analysis of a number of millions of fused populations of yeast protoplasts; for example, about 1 hour for analysis of millions of cells, of 1 hour for sorting submillions of cells to obtain thousands of fused protoplasts. About 1% of the population was sorted, and 1% of them was regenerated. At last the yield was some 10^<-4>.Fourth, cell fusion was ascertained by pulse-field agarose gel electrophoresis of chromosomes extracted from the cells and by measuring DNA content of the cells by flow cytometry with propidium iodide labelling of cells.