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

通过双荧光标记的弗洛姆细胞术从酵母原生质体中选择融合细胞

• 批准号: 01560128
• 负责人: KATSURAGI Tohoru
• 金额: $ 1.09万
• 依托单位: The University of Osaka Prefecture
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01560128/
• 关键词: Cell fusion of yeasts; Breeding of yeast; Cell sorter; Flow cytometry; Dual fluorescence labelling; Amylases, molecular breeding of; Alcohol fermentation; 蛍光標識; 酵母育種; 細胞膜蛍光染色; 核の蛍光染色(細胞の核)

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.

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。该模型分别是酵母糖化酵母和 Saccharomycopsis fibuligera 中的葡糖淀粉酶和 α-淀粉酶。使用的流式细胞仪是FACStar(Becton Dickinson免疫细胞计数系统公司)，其配备有细胞分选仪。首先，使用荧光分光光度计、微机辅助共焦激光显微镜和流式细胞仪筛选荧光染料。选择异硫氰酸荧光素异构体I和罗丹明6G，它们分别是标记细胞膜和线粒体的最佳选择。确定最佳标记条件并使用。其次，选择具有双荧光标记的融合细胞并在配备显微操作器的荧光显微镜下收集。获得少量融合细胞需要很长时间。第三，将流式细胞术引入上述双荧光标记的细胞分选中。研究了流式细胞仪和细胞分选仪的操作条件。我们花了很短的时间来分析数百万个融合的酵母原生质体群体；例如，分析数百万个细胞大约需要1小时，分选数百万个细胞以获得数千个融合原生质体大约需要1小时。大约1%的种群被分选，1%的种群被再生。最后产量约为10^-4。第四，通过对从细胞中提取的染色体进行脉冲场琼脂糖凝胶电泳并通过用细胞的碘化丙啶标记的流式细胞术测量细胞的DNA含量来确定细胞融合。