ISOLATION AND QUANTIFICATION OF CLASS-1 MHC GENE MUTANTS IN MOUSE T-CELLS BY IMMUNOSELECTION WITH A MAGNETIC CELL SORTER (MACS)

A method is described for determining the frequency of cells with a mu tation in an autosomal gene coding for a membrane protein. Using a mon oclonal antibody to H-2K(k) surface antigen and magnetic cell separati on (MACS) more than 10(4)-fold enrichment of the H-2K(k) negative popu lation was achieved, as tested with artificial mixtures containing a k nown number of antigen-negative cells. After a second magnetic sorting mutant frequencies as low as 10(-6) could be measured. The number of clonogenic mutants was evaluated by limiting dilution cloning and veri fication of the mutant phenotype by FACScan (flow cytometry) analysis in a representative number of clones. The spontaneous frequency of H-2 K(k) deficient mutants was 0.80 X 10(-6), and this increased after irr adiation with 6 Gy X rays to 3.38 x 10(-6) within the next 8 weeks. Ab out 50 mutant clones were screened for the presence of other class 1 a ntigens on the cell surface by FACScan analysis. All mutants continued to express other class 1 antigens.