Abstract: Abstract：Objective To construct Drosha-1 deficient 293 stable cells (293-siDrosha) and to study microRNA processing by using the established 293 stable cells. Methods The pSicoR-human Drosha1 plasmid DNAs were transfected into 293 cells and selected for neomycin resistant clones with 1200g/ml G418 for 2-3 weeks. The mRNA and protein expressions of Drosha were detected by RT-PCR，real-time PCR and Western blotting. The expressions of hsa-miR-491-5p in 293 cells, uncloned 293-siDrosha cells (293-siDrosha mixed) and the cloned 293-siDrosha cells (293-siDrosha #3 cells) were detected with poly(A) real-time RT-PCR. Results Compared with the control cells, the mRNA and protein expressions of human Drosha1 in cloned 293-siDrosha cells were dramatically decreased. The expression levels of hsa-miR-491-5p in the cloned 293-siDrosha cells have about 120 fold reduction as compared with that of the control cells. Conclusion 293-siDrosha stable cells were successfully constructed. The cloned 293-siDrosha cells may be useful as a model system for the study of microRNA functions.

Key words: siDrosha, microRNAs, 293 cells