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

研究了酵母融合原生质体的流式细胞术在酵母育种中的应用。模型分别为酵母菌糖淀粉酶和α -淀粉酶。使用的流式细胞仪为FACStar (Becton Dickinson Immunocytometry Systems Inc.)，配备细胞分选器。首先利用荧光分光光度计、微机辅助激光共聚焦显微镜和流式细胞仪对荧光染料进行筛选。选择异硫氰酸荧光素异构体I和罗丹明6G分别作为标记细胞膜和线粒体的最佳材料。确定了最佳贴标条件，并进行了后续应用。其次，选择具有双荧光标记的融合细胞，在配备微操仪的荧光显微镜下采集。需要很长时间才能获得少量的融合细胞。第三，将流式细胞术引入上述双荧光标记细胞分选中。研究了流式细胞仪和细胞分选仪的操作条件。花了很短的时间分析了数百万个酵母原生质体的融合群体；例如，大约1小时分析数百万个细胞，1小时分选亚数百万个细胞以获得数千个融合的原生质体。大约1%的种群被分类，1%的种群被再生。最后的产量约为10^<-4>。第四，对提取的细胞染色体进行脉冲场琼脂糖凝胶电泳，并对细胞进行碘化丙啶标记的流式细胞术测定细胞的DNA含量，确定细胞融合情况。

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.