Identification of human genomic clones coding the major histocompatibility antigens HLA-a2 and HLA-B7 by DNA-mediated gene transfer.
We have screened a large number of isolated human genomic clones that hybridize to a cloned HLA cDNA probe for their ability to direct the synthesis of HLA-A, -B, and -C surface antigens on mouse L cells following DNA-mediated gene transfer. The surface expression of human histocompatibility antigens, monitored by indirect immunofluorescence and the fluorescence-activated cell sorter, was examined at 60 hr after transfection and on hypoxanthine/aminopterin/thymidine-resistant (HATR) populations derived from cotransfer with the herpes simplex virus thymidine kinase gene. Two unique genomic clones designated JY B3.2 and JY 158, isolated from the human lymphoblastoid cell line JY (homozygous HLA-A2, -B7), were shown to contain gene sequences capable of directing expression of an HLA-A, -B, -C determinant. By using allo-specific antibodies, the gene products of these clones were identified as HLA-A2 and HLA-B7, respectively. HATR clonal populations isolated from cotransfections with these genomic clones displayed varying levels of surface HLA expression that correlated with the number of intact donor HLA sequences present in the cells. In general, these levels of expression were stable during 3 months in culture. This system provides a powerful tool for the study of human surface antigen gene structure, expression, and function on a mouse cell background.
D000954:Antigens, Surface / Q000235:genetics; D002469:Cell Separation; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005796:Genes; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D008285:Major Histocompatibility Complex; D014170:Transformation, Genetic
D000954:Antigens, Surface; D006680:HLA Antigens