Herzenberg L A, Hsu C, Alberti S, Kavathas P
Med Oncol Tumor Pharmacother. 1984;1(4):219-24. doi: 10.1007/BF02934526.
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.
为便于克隆细胞表面分子的基因，我们用胸苷激酶（TK）基因与人或小鼠的总DNA共转染LTK-小鼠成纤维细胞。通过在HAT培养基中生长筛选出的TK+细胞，用与一种或另一种膜分化抗原或受体结合的荧光染料偶联单克隆抗体或其他荧光配体进行染色。我们使用荧光激活细胞分选仪（FACS）分离表达这些分子的荧光转染子。对于某些抗原，会发生自发基因扩增。通过FACS分选和再生长的重复循环，我们获得了高表达克隆。然后，我们使用选定的cDNA探针筛选带有cDNA插入片段的噬菌体，分离出cDNA和基因组克隆。除了源自滋养层的绒毛膜癌细胞系的DNA不能转染Leu-2外，几乎任何组织来源的DNA对各种分子的转染效果都同样良好。
