Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. This study demonstrates that shRNAs can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or transcribed from RNA polymerase III promoters in vivo, enabling the creation of continuous cell lines or transgenic animals with stable and heritable gene silencing. RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a response to double-stranded RNA (dsRNA), which induces gene silencing. RNAi is a conserved regulatory mechanism present in many eukaryotic organisms. Recent studies show that endogenous triggers of gene silencing act through RNAi machinery to regulate gene expression. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors, processed into active 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. shRNAs can be designed to mimic stRNAs and induce gene silencing in mammalian cells. The study shows that shRNAs can be used to induce gene silencing in mammalian cells, with potency similar to siRNAs. shRNAs can be synthesized in vitro using T7 RNA polymerase, and their effectiveness in inducing gene silencing was demonstrated in various mammalian cell lines. The study also shows that shRNAs can be transcribed in vivo from RNA polymerase III promoters, enabling stable gene silencing in mammalian cells. The results suggest that shRNAs can be used to create continuous cell lines and transgenic animals with stable gene silencing. The study highlights the potential of shRNAs as a tool for gene function studies in mammalian cells.Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. This study demonstrates that shRNAs can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or transcribed from RNA polymerase III promoters in vivo, enabling the creation of continuous cell lines or transgenic animals with stable and heritable gene silencing. RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a response to double-stranded RNA (dsRNA), which induces gene silencing. RNAi is a conserved regulatory mechanism present in many eukaryotic organisms. Recent studies show that endogenous triggers of gene silencing act through RNAi machinery to regulate gene expression. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors, processed into active 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. shRNAs can be designed to mimic stRNAs and induce gene silencing in mammalian cells. The study shows that shRNAs can be used to induce gene silencing in mammalian cells, with potency similar to siRNAs. shRNAs can be synthesized in vitro using T7 RNA polymerase, and their effectiveness in inducing gene silencing was demonstrated in various mammalian cell lines. The study also shows that shRNAs can be transcribed in vivo from RNA polymerase III promoters, enabling stable gene silencing in mammalian cells. The results suggest that shRNAs can be used to create continuous cell lines and transgenic animals with stable gene silencing. The study highlights the potential of shRNAs as a tool for gene function studies in mammalian cells.