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Use of fluorescence-activated cell sorter to isolate mutant mammalian cells deficient in an internal protein,
Abstract:
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.
Insights
Researchers developed a new method to isolate cells lacking specific internal proteins. This technique uses a fluorescence-activated cell sorter to efficiently identify and select dihydrofolate reductase-deficient mutants, improving cell screening capabilities.
Area of Science:
- Cell Biology
- Molecular Genetics
- Biochemistry
Background:
- Dihydrofolate reductase (DHFR) is crucial for nucleotide synthesis.
- Methotrexate is a substrate analog that inhibits DHFR.
- Selecting for DHFR-deficient mutants is important for various genetic studies.
Purpose of the Study:
- To develop an efficient method for isolating Chinese hamster ovary (CHO) cell mutants deficient in dihydrofolate reductase (DHFR).
- To utilize a fluorescence-activated cell sorter (FACS) for mutant selection based on ligand binding.
- To demonstrate the applicability of FACS for isolating mutants with altered internal protein expression.
Main Methods:
- Chinese hamster ovary (CHO) cells were mutagenized.
- Cells were screened for their inability to bind a fluorescent methotrexate derivative using FACS.
- Multiple rounds of cell sorting and regrowth were performed to enrich for mutant cells.
Main Results:
- A fluorescence-activated cell sorter successfully isolated nonfluorescent DHFR-deficient mutants.
- The mutant cell frequency was increased from 10^-5 to over 0.9 through iterative sorting.
- The method demonstrated high efficiency in selecting for cells with altered DHFR expression.
Conclusions:
- FACS is a powerful tool for isolating mutants based on the binding of fluorescent ligands.
- This method is broadly applicable for selecting gene products that bind fluorescent molecules tightly.
- The technique allows for the screening of large cell populations and selection of partially expressed phenotypes.