Transfection and cloning of genes for membrane antigens using the FACS.
In order to facilitate cloning of genes for cell surface molecules, we cotransfected LTK- mouse fibroblasts with thymidine kinase (TK) genes and total human or mouse DNA. TK+ cells, selected by growth in HAT medium, were stained with fluorochrome conjugated monoclonal antibodies or other fluorescent ligands which bind to one or another membrane differentiation antigen or receptor. We isolated fluorescent transfectants expressing these molecules using a fluorescence activated cell sorter (FACS). For some antigens, spontaneous gene amplification occurred. By repeated cycles of FACS sorting and regrowth we obtained high expressing clones. We then isolated cDNA and genomic clones using selected cDNA probes to screen phage with cDNA inserts. DNA from virtually any tissue source transfected equally well for the various molecules except for DNA from a trophoblast derived choriocarcinoma cell line which did not transfect for Leu-2.
D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface / Q000235:genetics*; D002469:Cell Separation; D003001:Cloning, Molecular; D004247:DNA; D005434:Flow Cytometry; D005784:Gene Amplification; D006801:Humans; D007739:L Cells / Q000276:immunology; D051379:Mice; D013937:Thymidine Kinase / Q000235:genetics; D014170:Transformation, Genetic
D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D004247:DNA; D013937:Thymidine Kinase