부산대학교 도서관

We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-rasm gene and an N-rasm suppressor, respectively. Retroviral vectors were produced in which human N-rasm (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-rasm was coexpressed with RFP. N-Rasm suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-rasm suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-rasmsuppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-rasm suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-rasm-induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.Cancer Gene Therapy (2003) 10, 745-754. doi:10.1038/sj.cgt.7700603 [ABSTRACT FROM AUTHOR]