A DNA vector-based RNAi technology has been developed to suppress gene expression in mammalian cells. This method allows the synthesis of small interfering RNAs (siRNAs) from DNA templates in vivo, leading to efficient inhibition of endogenous gene expression. The technology utilizes RNA polymerase III (Pol III) to generate small, noncoding transcripts with defined 3' ends, which are structurally similar to siRNAs synthesized in vitro. This approach enables the specific suppression of gene expression in mammalian cells, as demonstrated by the inhibition of multiple endogenous genes, including lamin A/C, cyclin-dependent kinase-2 (cdk-2), and DNA methyltransferase-1 (dnmt-1). The study shows that siRNAs synthesized from DNA templates in vivo can effectively inhibit the expression of both transfected and endogenous genes. For example, in HeLa cells, the expression of the green fluorescent protein (GFP) was significantly reduced when transfected with a plasmid containing a DNA template for siRNA targeting GFP. Similarly, the expression of lamin A/C, cdk-2, and dnmt-1 was inhibited in transfected cells. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells. The technology has broad applications for studying gene function in mammalian cells and may be adapted for long-term stable inhibition or inducible siRNA systems. This approach provides a powerful tool for gene function analysis and could facilitate the use of RNAi in cell culture and vertebrate animals. The study also demonstrates that this method is effective across various cell lines, including HeLa, H1299, C-33A, and U-2 OS cells. The results indicate that this DNA vector-based RNAi technology can achieve robust, near-complete inhibition of endogenous gene expression, making it a valuable tool for mammalian gene function studies.A DNA vector-based RNAi technology has been developed to suppress gene expression in mammalian cells. This method allows the synthesis of small interfering RNAs (siRNAs) from DNA templates in vivo, leading to efficient inhibition of endogenous gene expression. The technology utilizes RNA polymerase III (Pol III) to generate small, noncoding transcripts with defined 3' ends, which are structurally similar to siRNAs synthesized in vitro. This approach enables the specific suppression of gene expression in mammalian cells, as demonstrated by the inhibition of multiple endogenous genes, including lamin A/C, cyclin-dependent kinase-2 (cdk-2), and DNA methyltransferase-1 (dnmt-1). The study shows that siRNAs synthesized from DNA templates in vivo can effectively inhibit the expression of both transfected and endogenous genes. For example, in HeLa cells, the expression of the green fluorescent protein (GFP) was significantly reduced when transfected with a plasmid containing a DNA template for siRNA targeting GFP. Similarly, the expression of lamin A/C, cdk-2, and dnmt-1 was inhibited in transfected cells. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells. The technology has broad applications for studying gene function in mammalian cells and may be adapted for long-term stable inhibition or inducible siRNA systems. This approach provides a powerful tool for gene function analysis and could facilitate the use of RNAi in cell culture and vertebrate animals. The study also demonstrates that this method is effective across various cell lines, including HeLa, H1299, C-33A, and U-2 OS cells. The results indicate that this DNA vector-based RNAi technology can achieve robust, near-complete inhibition of endogenous gene expression, making it a valuable tool for mammalian gene function studies.