Transfection of HLA genes using genomic DNA.
Mouse L cells expressing HLA genes are potentially useful for producing and analyzing monoclonal antibodies (mAb) to HLA molecules. This paper describes the preparation of transfectants using uncloned human DNA and three methods to isolate the HLA-expressing transfectants. Transfectant libraries were made by cotransfecting mouse thymidine kinase (tk)-deficient L cells with a calcium phosphate precipitate containing genomic DNA and tk plasmid DNA. Transfectants expressing HLA genes were isolated using these methods: immunomagnetism, replicate-plating combined with cellular enzyme-linked immunoassay, and sorting using a fluorescence-activated cell sorter. Two HLA-A2 transfectant were isolated using immunomagnetism, two HLA-A24 transfectants by replicate plating, and one HLA-Bw60 transfectant by FACS. However, no transfectants were isolated that stably expressed class II genes. The class I transfectants have been useful in characterizing several locally prepared mAbs which bind to monomorphic determinants on class I HLA molecules. Two of the transfectant lines, one expressing HLA-A2 (8001) and the other HLA-A24 (8008), have been included in the collection of lines distributed for use in the Eleventh International Histocompatibility Workshop.
Drover
S
S
Immunology Laboratory, Faculty of Medicine, Memorial University of Newfoundland, St. John's Canada.
Marshall
W H
WH
D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006680:HLA Antigens / Q000235:genetics* / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D014162:Transfection
D000911:Antibodies, Monoclonal; D006680:HLA Antigens; D004247:DNA