|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1990 : ¥300,000 (Direct Cost : ¥300,000)
Fiscal Year 1989 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.