EFFICIENT GENE ACTIVATION SYSTEM ON MAMMALIAN-CELL CHROMOSOMES USING RECOMBINANT ADENOVIRUS PRODUCING CRE RECOMBINASE

To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cr e was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excis ional deletion of the neo gene. CV1 cell lines bearing the switching u nit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovi rus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populati ons reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the syst em offers efficient activation of genes introduced into cell chromosom es and that the organization of the reporter units are important for e fficiency and duration of the activated gene expression.