Use of fluorescence-activated cell sorter to isolate mutant mammalian cells deficient in an internal protein, dihydrofolate reductase.
Dihydrofolate reductase-deficient mutants of Chinese hamster ovary cells have been selected based on their inability to bind a fluorescent derivative of methotrexate, a substrate analog. Nonfluorescent mutant cells were isolated from mutagenized populations using a fluorescence-activated cell sorter. After multiple rounds of sorting plus regrowth, the mutant cell frequency was increased from an initial 10(-5) to greater than 0.9. This use of the cell sorter to isolate mutants deficient in an internal protein should be applicable to any gene product that is able to bind a fluorescent ligand tightly. The method has the advantage of allowing the screening of large numbers of cells and of selecting for partially expressed phenotypes.
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D009154:Mutation; D010053:Ovary; D013762:Tetrahydrofolate Dehydrogenase / Q000172:deficiency* / Q000235:genetics
D013762:Tetrahydrofolate Dehydrogenase