RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells

TLDR

Duplexes of 21‑nt RNAs, known as short‑interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. The study demonstrates that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, offering an economical alternative to chemical synthesis. The authors use T7 RNA polymerase–driven in vitro transcription to generate siRNAs and short hairpin siRNAs that act like duplexes to inhibit gene expression in a sequence‑specific manner. The authors demonstrate that U6‑driven hairpin siRNAs effectively silence target genes—including a neuron‑specific β‑tubulin during differentiation—while mismatches enhance strand selectivity, indicating a rapid, versatile RNAi tool with therapeutic potential.

Abstract

Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific β-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.