An approach to the cloning of cell surface protein genes. Selection by cell sorting of mouse L-cells that express HLA or 4F2 antigens after transformation with total human DNA.
We describe an approach to the cloning of cell surface proteins that is independent of messenger RNA isolation. Mouse Ltk- cells are cotransformed with the thymidine kinase gene from Herpes Simplex Virus and total human DNA. Transformants expressing the human surface antigens of interest are isolated by two selection steps, consisting of treatment with hypoxanthine/aminopterin/thymidine and fluorescence-activated cell sorting. Using this procedure, we isolated seven transformants expressing HLA-A,B,C antigens and 12 transformants expressing the 4F2 antigen. We have so far failed to identify any OKT-10 antigen expressing L-cell transformants. Three independent secondary 4F2 transformants were obtained after identical cotransformation of fresh Ltk- cells with DNA from primary transformants. Analysis of their genome by hybridization with human DNA revealed a shared set of human restriction fragments in all three cell lines. This 32 X 10(3) base-pair segment of DNA codes for the human 4F2 antigen, thereby offering the opportunity to clone the gene. To substantiate this hypothesis, we analyzed the seven HLA-expressing cell lines, and we found that all of them had acquired an HLA-coding sequence concomitant to its expression.
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D008040:Genetic Linkage; D006168:Guinea Pigs; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D007739:L Cells; D014170:Transformation, Genetic
D000954:Antigens, Surface; D006680:HLA Antigens