An immunocytochemical screening of human-mouse cell hybrid colonies expressing a specific human gene

An immunocytochemical method has been devised which allows the screening of a large number of human x mouse cell hybrid colonies for the retention of a specific human chromosomal gene and the presence of its translation product. The glycolytic enzyme phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was chosen as a marker which is known to be controlled by the gene on human chromosome 19. The technique involves three steps: (i) immobilization of growing cell colonies in agar gel containing antibody that specifically reacts with human-type PGI; (ii) lysis of the embedded cells with Triton X-100 to release enzyme antigens and precipitate as an immune complex; and (iii) visualization of the antibody-fixed enzymes by histochemical activity staining. Human PGI activity released from a colony consisting of as few as eight cells generated an adequate signal. Variation of intensity was noticed and attributed to gene dosage in individual cells. The percentage of human PGI-positive colonies in each of nine independent hybrid lines estimated by this method generally paralleled the frequency of retention of human chromosome 19 determined by conventional karyotyping. The technique can be applied to many other markers and be used as "a half-selection" system in combination with the "replica plating" method.