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

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: Frog Prince; RNA interference; Sleeping Beauty; nonviral gene transfer; short hairpin RNA; stable gene knockdown; transposon-based gene delivery.

Figure 1.

Schematic of Frog Prince-based shRNA vectors. (A) The parental FP transposon contains the neomycin resistance gene (NEO) driven by the SV40 promoter (SV40) and followed by the SV40 polyadenlyation signal (SV40-pA) between the left and right flanking terminal inverted repeats (IR). (B) The H1 promoter expression cassette from pSUPER was subcloned between the polyadenylation signal and the right IR to generate pFP/Neo-H1.

Figure 2.

Stable FP-shRNA-mediated knockdown of EGFP in pooled HeLa colonies. The EGFP clonal HeLa cell line H38-3 was cotransfected with the indicated shRNA expressing plasmid plus either a transposase source or a neomycin expression plasmid. Transfected cells were selected with G418 for two weeks, and resistant colonies were harvested and pooled together. EGFP expression was analyzed by FACS. The mean EGFP expression of the H38-3 parental line was arbitrarily set to 100%. The respective overlaid FACS profiles are shown in the inset.

Figure 3.

Comparison of efficiencies of colony formation and EGFP knockdown with pSUPER- versus pFP-based shRNA vectors. The EGFP clonal HeLa line H38-3 was cotransfected with (A) pSUPER/EGFP4 and pFP/Neo, or (B) pFP/Neo-H1/EGFP4 and pFV-FP. Two days after transfection, the cells were diluted and plated into 96-well plates. After two weeks under G418 selection, EGFP expression was analyzed for resistant colonies in each well with a plate reader. Colonies were then fixed, stained and counted. The EGFP expression level per colony was calculated and graphically displayed using the Treeview program. Each square pixel represents a well and is color-coded. Black represents empty wells, while the shades of green reflect the level of EGFP expression per colony, with dark meaning a low level and bright meaning a high level.

Figure 4.

Efficiency of FP-shRNA-mediated knockdown of Ku70 in HeLa cells assayed by Western hybridization and Sleeping Beauty transposition. (A) Western hybridizations of nuclear protein extracts prepared from HeLa cells transiently transfected with the plasmid constructs shown above the lanes. Nuclear extracts from each transfection were blotted and hybridized with a Ku70 antibody (upper panel) or with an actin antibody (lower panel). (B) Efficiency of SB transposition in transgenic cell clones expressing shRNAs against Ku70. The indicated numbers of colonies (n) from each transfection were picked and expanded under selection. A transposition assay using a zeocin resistance gene-marked SB transposon and a SB transposase helper plasmid was performed in each clone. After two weeks under zeocin selection, resistant colonies were stained and counted. The efficiency of SB transposition in each picked clone was grouped into five categories: 0, 1-25, 25-50, 50-75 and 75-100% reduction, relative to the level of SB transposition in the pSUPER control clones.