Expression of the human monocyte membrane antigen gp55 by murine fibroblasts after DNA-mediated gene transfer

Abstract

Abstract Human DNA sequences that contain the gene encoding gp55, a cell surface glycoprotein expressed exclusively on mature human monocytes and monocytic leukemia cells, were isolated in a mouse genetic background. DNA from mature human monocytes was cotransfected with DNA from a molecularly cloned feline sarcoma virus containing the v-fms oncogene into NIH-3T3 cells. Transformed mouse fibroblasts that expressed gp55, based on their reactivity with the MY4, B44.1, or LeuM3 monoclonal antibodies, were selected by fluorescence-activated cell sorting. Regardless of which antibody was used for selection, equivalent binding of all three antibodies was observed for positive transformants. Secondary and tertiary mouse cell transformants were obtained after additional rounds of transfection and cell sorting with the use of DNA from primary and then secondary transformants. Southern blot analysis of the cellular DNA from two independently derived tertiary subclones revealed a limited complement of human sequences, thus indicating that the gene encoding gp55 is included in fewer than 50 kilobases of human DNA. Independently derived tertiary subclones displayed concordant patterns of reactivity with 13 monocyte-specific monoclonal antibodies, thus indicating that each recognized an epitope on the product (gp55) of a single human gene. The 55-kilodalton cell surface polypeptide was specifically immunoprecipitated with a representative monoclonal antibody, 26if, from lysates of enzymatically radioiodinated peripheral blood monocytes and tertiary transformants. We conclude that gp55 is highly immunogenic and that a large number of independently derived monoclonal antibodies specific for human monocytes react with epitopes on this one molecule.