L1 retrotransposon-mediated stable gene silencing

Abstract

RNA interference (RNAi) is widely used for functional studies and has been proposed as a potential therapeutic agent. Current RNAi systems are largely efficient, but have limitations including transient effect, the need for viral handling and potential insertional mutations. Here, we describe a simple L1 retrotransposon-based system for the delivery of small interfering RNA (siRNA) and stable silencing in human cells. This system demonstrated long-term siRNA expression and significant reduction in both exogenous and endogenous gene expression by up to 90%. Further characterization indicated that retrotransposition occurred in a controlled manner such that essentially only one RNAi-cassette was integrated into the host genome and was sufficient for strong interference. Our system provides a novel strategy for stable gene silencing that is easy and efficient, and it may have potential applications for ex vivo and in vivo molecular therapy.

Figure 1

L1 retrotransposon-based RNAi system. (A) Mechanism of L1 retrotransposition. (B) Strategy for L1-based RNAi. A selectable neo-cassette for retrotransposition is inserted in the 3′-UTR of L1_RP, followed by an RNAi-cassette composed of the H1 promoter, the 19 bp sense and antisense sequences of a specific gene separated by a 9 bp loop sequence and a pol III terminator of six thymidines. Abbreviations: pA, polyadenylation signal; NEO, neo gene; SD, splicing donor site; and SA, splicing acceptor site. Arrows indicate transcription start sites. (C) Structure of L1-based RNAi vector (pL1-Silencer) and expressed shRNA. (D) L1-based RNAi system. Transfection of pL1-Silencer into cultured cells is followed by the transcription of L1-RNAi sequence, its integration into the host genome and stable siRNA expression.

Figure 2

Inhibition of exogenous GFP expression using L1-based RNAi system. (A) Real-time RT–PCR results of GFP mRNA expression in stable HeLa lines expressing control or GFP-targeted siRNA (n = 6). (B) Western-blot analysis of GFP expression in control or knockdown clones. (C) Typical FACS dot plots of GFP expression in control and knockdown clones. (D) Normalized GFP fluorescence in control and knockdown clones by FACS (n = 4).

Figure 3

Inhibition of endogenous GAPDH expression using L1-based RNAi system. (A) Real-time RT–PCR results of GAPDH mRNA expression in stable HeLa lines expressing control, GFP-targeted or GAPDH-targeted siRNA (n = 6). (B) Western-blot analysis of GAPDH protein expression in control or GAPDH knockdown clones. (C) Immunofluorescent staining of GAPDH (green) and cytokeratin (red) in control and knockdown cells. Nucleus is counterstained with DAPI (blue).

Figure 4

Northern-blot analysis of siRNA expression in GAPDH knockdown cells. Upper panel (from left to right): lane 1, 19 nt probe containing the sense strand sequence of GAPDH siRNA as a molecular weight marker (MW), which was also used as the probe to detect antisense siRNA in the test samples; lane 2: wild-type HeLa (wt); lanes 3–6: four GAPDH knockdown lines (#1, 2, 3 and 4). Position of antisense siRNA is indicated. Note the inverse relationship of siRNA expression seen here with GAPDH mRNA and protein expression (Figure 3A and B). Lower panel: gel post-electrophoresis. Positions of 5.8S, 5S and tRNAs are indicated.

Figure 5

G418 resistant clones are derived from retrotransposition. (A) PCR strategy to characterize retrotransposition of tagged L1. The primer pair (F and R) produces a PCR product of 1679 bp before or of 775 bp post retrotransposition of tagged L1. (B) Electrophoresis of the PCR product of genomic DNA from knockdown clones, plus controls with no DNA (−DNA), wild-type HeLa DNA (Mock) or pL1-Silencer plasmid (Plus Intron). MW, molecular weight marker. Arrows indicate the positions of expectant bands.

Figure 6

L1 retrotransposition occurs in a controlled manner. (A) Retrotransposition frequency is linear in cultured cells up to day 7 post-transfection as revealed by FACS (n = 4). JM111, a negative control of L1 with eliminated retrotransposition activity. (B) Retrotransposition frequency reaches a plateau around day 7 post-transfection (n = 4). (C) Real-time PCR strategy to characterize retrotransposition of GFP-cassette tagged L1. Arrows, primers; bar, Taqman probe. (D) Retrotransposition frequency of exogenously introduced L1_RP, L1.3 and JM111 in HeLa on day 7 post-transfection as revealed in parallel by real-time PCR (upper panel) in which data are represented as copy numbers of intact GFP per 1000 GAPDH and by FACS (lower panel) in which data are represented as percentage of GFP positive cells among transfected cells.