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. 2004 May 11;101(19):7347-51.
doi: 10.1073/pnas.0402107101. Epub 2004 Apr 30.

CRE recombinase-inducible RNA interference mediated by lentiviral vectors

Gustavo Tiscornia  1 , Vinay Tergaonkar, Francesco Galimi, Inder M Verma
Affiliations

CRE recombinase-inducible RNA interference mediated by lentiviral vectors

Gustavo Tiscornia et al. Proc Natl Acad Sci U S A. .

Abstract

Recently, several systems designed to trigger RNA interference by using small hairpin RNA driven by polymerase III promoters have been described. Here, we report a lentiviral-mediated small interfering RNA delivery system that can be induced by CRE recombinase. The system consists of a lentiviral vector carrying a mouse U6 promoter that is separated from a small hairpin RNA by a random DNA stuffer sequence flanked by modified loxP sites. The silencing cassette is not expressed until activated by addition of CRE recombinase delivered by a lentiviral vector. We have used this system to show specific down-regulation of GFP and two endogenous genes (the tumor suppressor p53 and the NF-kappaB transcription factor subunit p65) in vitro. Furthermore, down-regulation of both p53 and p65 resulted in the expected effect on downstream genes and cellular phenotype. We foresee multiple applications of this system both in vitro and in vivo to down-regulate specific targets in a tissue-specific and localized manner.

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Figures

Fig. 1.
Fig. 1.
Diagram of lentiviral constructs (integrated proviral form) used in this study. (A) LV-shGFP-WT: lentiviral vector carrying a wild-type silencing cassette. The mU6 promoter consists of three elements: distal sequence element, PSE, and TATA box. The circled numbers (1, 2, and 3) indicate sites where unsuccessful attempts were made to introduce loxP sites. (B) LV-shRNA-OFF: the introduction of a DNA stuffer sequence (LacZ) flanked by modified loxP sites (loxP*). loxP* is a loxP site containing two mutations in the 8-bp spacer region (GCATACAT to GTATAAA), resulting in a modified loxP site (loxP*) with a spacer that resembles a U6 TATA box. The presence of a stuffer fragment puts the promoter in an OFF configuration. The promoter will be silent until CRE recombinase excises the fragment, leaving one loxP* site (LV-shRNA-ON) (C) and switching the silencing cassette from OFF to ON. The shRNA is thus expressed, leading to siRNA synthesis and target shutdown. (D) LV-shRNA+GFP: lentiviral construct allowing the delivery of both a silencing cassette and a GFP marker. PPT, polypyrimidine tract; WPRE, woodchuck hepatitis virus posttranscriptional response element; all viral elements are shown on a white background; DSE, distal sequence element; TATA, mU6 TATA box; loxP*, modified loxP site with spacer resembling mU6 TATA box; all mU6 promoter elements are shown on a violet background, except for T5 (pol III transcription termination signal), which is shown on a red background. The stuffer fragment (randomly chosen 1-kb region of LacZ) is shown on a yellow background and is present only in LV-shRNA-OFF. The GFP marker in LV-shRNA is shown on a green background. shRNA hairpins are shown in black.
Fig. 2.
Fig. 2.
(A) 293T cells expressing two copies of GFP were infected with different lentiviral constructs, with mois as shown in B. Vector titers were determined by p24 ELISA and by TaqMan real-time PCR determination of transduced proviral genomes. On average, the vector preparations were 1.5 E4 particles per nanogram of p24. Cells were cultured for 9 days, and GFP expression levels were quantitated by FACS analysis. GFP expression levels were unaffected by infection with LV-CRE alone (Aa). Infection with LV-shGFP-OFF in the absence of LV-CRE resulted in no change in GFP levels (Ab), whereas LV-shGFP-WT and LV-shGFP-ON were equally effective in down-regulating the target (A c and d). Coinfection of LV-CRE (moi = 70) and LV-shGFP-OFF (moi = 100) resulted in efficient down-regulation of GFP expression (Ae). (B) FACS analysis composite. Coinfection of LV-CRE (moi = 70) with decreasing mois of LV-siGFP-OFF (moi = 60, 20, or 10) resulted in decreased silencing of GFP (data not shown). Similarly, decreasing of LV-CRE (moi = 70, 35, 7, and 3) while keeping LV-shGFP-OFF constant (moi = 100) diminished silencing (data not shown).
Fig. 3.
Fig. 3.
Regulated knockdown of endogenous genes. (A) Wild-type murine embryo fibroblasts (MEFs) were infected with mois of 50 (lane 3), 100 (lane 4), 200 (lane 5), and 400 (lanes 6 and 7) of LV-shp65-OFF, with (lanes 3-6) or without (lane 7) coinfection with LV-CRE at an moi of 700. Cells were cultured for 5 days, and lysates were analyzed for the indicated markers by Western blot analysis. LV-shp65-OFF used in combination with LV-CRE (but not when used alone) resulted in highly decreased levels of p65 and downstream targets. Actin was used as a loading control, and p50 and a nonspecific band (NS) are included to show specificity. (B) Tumor necrosis factor α-induced apoptosis. MEFs infected with LV-shp65-WT +GFP (moi = 600) were used as a positive control (lane 2), and LV-shp65-OFF (moi = 400) in combination with LV-CRE (moi = 700) showed increased sensitivity to apoptosis after being assayed as described in Methods.(C) Wild-type MEFs were infected with mois of 25 (lane 4), 125 (lane 5), 250 (lane 6), and 500 (lanes 7 and 8) of LV-shp53-OFF, with (lanes 4-7) or without (lane 8) coinfection with LV-CRE at moi 700. Cells were cultured for 5 days and either left untreated (lane 1) or treated with 0.4 μg/ml doxorubicin (lanes 2-8) and assayed by Western blotting. Cells infected with LV-shp53-WT+GFP (moi = 600, lane 3) were used as a positive control. Increased mois of LV-shp53-OFF (moi = 500) in combination with LV-CRE (moi = 700) resulted in increasingly stronger down-regulation of p53 and downstream targets (lanes 4-7). Actin was used as a loading control, and a nonspecific band is included to show specificity.
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References

    1. Wilson, J. A. & Richardson, C. D. (2003) Curr. Opin. Mol. Ther. 5, 389-396. - PubMed
    1. Zhao, L. J., Jian, H. & Zhu, H. (2003) Gene 316, 137-141. - PubMed
    1. Tomar, R. S., Matta, H. & Chaudhary, P. M. (2003) Oncogene 22, 5712-5715. - PubMed
    1. Sorensen, D. R., Leirdal, M. & Sioud, M. (2003) J. Mol. Biol. 327, 761-766. - PubMed
    1. Somasundaram, K. (2003) Cancer Biol. Ther. 2, 211-212. - PubMed

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