This article describes a DNA vector-based RNA interference (RNAi) technology that allows the synthesis of small interfering RNAs (siRNAs) from DNA templates in mammalian cells. The authors developed a strategy using the RNA polymerase III (Pol III) promoter to generate siRNAs with the required structural features for effective gene silencing. They demonstrated that this approach can efficiently inhibit both transfected and endogenous genes in multiple cell lines, including HeLa, U-2 OS, H1299, and C-33A cells. The siRNAs synthesized from DNA templates were shown to specifically and robustly reduce the expression of target genes, as evidenced by immunofluorescence microscopy and Western blotting. The study highlights the broad applicability of this DNA vector-based RNAi technology for gene function analysis in mammalian cells and potentially in vertebrate animals.This article describes a DNA vector-based RNA interference (RNAi) technology that allows the synthesis of small interfering RNAs (siRNAs) from DNA templates in mammalian cells. The authors developed a strategy using the RNA polymerase III (Pol III) promoter to generate siRNAs with the required structural features for effective gene silencing. They demonstrated that this approach can efficiently inhibit both transfected and endogenous genes in multiple cell lines, including HeLa, U-2 OS, H1299, and C-33A cells. The siRNAs synthesized from DNA templates were shown to specifically and robustly reduce the expression of target genes, as evidenced by immunofluorescence microscopy and Western blotting. The study highlights the broad applicability of this DNA vector-based RNAi technology for gene function analysis in mammalian cells and potentially in vertebrate animals.