Journal of RNAi and Gene Silencing (October 2005), 1(2), 97-104

New Methods and Technologies

Frog Prince transposon-based RNAi vectors mediate efficient gene knockdown in human cells

Christopher D Kaufman †, Zsuzsanna Izsvák †‡, Andrea Katzer † and Zoltán Ivics †*

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

† 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

© Copyright Christopher D Kaufman et al

(Received 07 June 2005; Revised 04 July 2005; Accepted 06 July 2005)