RNA



METHODS

Functional siRNA expression from transfected PCR products


DANIELA  CASTANOTTO  a1, HAITANG  LI  a1 and JOHN J.  ROSSI  a1 c1
a1 Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA

Abstract

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. In mammalian systems, the sequence-specific RNAi effect has been observed by expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. We describe here a facile PCR based strategy for rapid synthesis of siRNA expression units and their testing in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in functional expression of siRNAs. This approach should prove useful for identification of optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.

(Received August 7 2002)
(Revised August 14 2002)
(Accepted August 29 2002)


Key Words: message down-regulation; Pol III expression; short hairpin RNAs; small interfering RNAs.

Correspondence:
c1 Reprint requests to: John J. Rossi, Division of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010, USA; e-mail: jrossi@bricoh.edu.