New Methods and Technologies J RNAi Gene Silenc (October 2005), 1(2), 97-104 doi: jrgsxx Published online: 02 September 2005 Full Text: (html | pdf ~348kb | refs) Frog Prince transposon-based RNAi vectors mediate efficient gene knockdown in human cells Christopher D Kaufman †, Zsuzsanna Izsvák †‡, Andrea Katzer † and Zoltán Ivics †* † Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany ‡ Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Hungary *Correspondence to: Zoltán Ivics, Email: zivics@mdc-berlin.de, Tel: +49 30 9406 2546, Fax: +49 30 9406 2547 Received: 07 June 2005, Revised: 04 July 2005, Accepted: 06 July 2005 © Copyright The Authors ----------------------------------------------------------------------------------------------------------------- ABSTRACT We have developed a stable RNA interference (RNAi) delivery system that is based on the Frog Prince transposable element. This plasmid-based vector system combines the gene silencing capabilities of H1 polymerase III promoter-driven short hairpin RNAs (shRNA) with the advantages of stable and efficient genomic integration of the shRNA cassette mediated by transposition. We show that the Frog Prince-based shRNA expressing system can efficiently knock down the expression of both exogenous as well as endogenous genes in human cells. Furthermore, we use the Frog Prince-based system to study the effect of knockdown of the DNA repair factor Ku70 on transposition of the Sleeping Beauty transposon. Transposon-mediated genomic integration ensures that the shRNA expression cassette and a selectable marker gene within the transposon remain intact and physically linked. We demonstrate that a major advantage of our vector system over plasmid-based shRNA delivery is both its enhanced frequency of intact genomic integration as well as higher target suppression in transgenic human cells. Due to its simplicity and effectiveness, transposon-based RNAi is an emerging tool to facilitate analysis of gene function through the establishment of stable loss-of-function cell lines. KEYWORDS: RNA interference, short hairpin RNA, Frog Prince, Sleeping Beauty, nonviral gene transfer, stable gene knockdown, transposon-based gene delivery ----------------------------------------------------------------------------------------------------------------- Privacy | Disclaimer |