RAPID DETECTION OF HOMOLOGOUS RECOMBINANTS IN NONTRANSFORMED HUMAN-CELLS

Gene targeting is a technique by which a preselected site in the genom e of a living cell fan be modified by inserting, deleting, or exchangi ng DNA sequences. The application of this technology to cells with a l imited life-span, such as nontransformed human somatic cells, requires the development of simplified and efficient procedures to allow the i solation of correctly modified cells from the much larger pool of rand om integrants. The current study describes the development of a widely applicable strategy for detecting homologous recombinants in human ce lls by using an ELISA-based screen, When this system is used accuratel y targeted clones can be detected with high efficiency as soon as 14 d ays following transfection. Data are presented demonstrating the utili ty of this detection system in isolating targeted recombinants at the beta(2)-microglobulin locus in both human retinal pigmented epithelial cells and human keratinocytes.