Kavathas P, Sukhatme V P, Herzenberg L A, Parnes J R
Proc Natl Acad Sci U S A. 1984 Dec;81(24):7688-92. doi: 10.1073/pnas.81.24.7688.
We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.
我们报告了通过转染、荧光激活细胞分选和消减cDNA杂交相结合的方法，分离编码人T淋巴细胞细胞表面分化抗原Leu-2（T8）的基因组和cDNA克隆。我们用来自表达并扩增人Leu-2基因的小鼠L细胞转染子的mRNA构建了一个cDNA文库。我们通过用来自第二个扩增的Leu-2转染子的选定cDNA探针进行筛选，鉴定出Leu-2 cDNA克隆。该探针包含未与L细胞mRNA杂交而去除的cDNA种类。一个Leu-2 cDNA克隆被用于分离一个基因组克隆。用该克隆的DNA进行转染产生了大量的Leu-2转染子。这种方法可用于克隆编码其他细胞表面分子的基因。
