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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Published: June 21st, 2013

DOI:

10.3791/50316

1Department of Pharmacology, Rush University, 2Pulmonary and Critical Care Department, Rush University
* These authors contributed equally

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Naked siRNA cannot penetrate the cell membrane, being negatively charged, and it is easily degraded by enzymes in blood, tissues, and cells. Even with recently structural modifications to improve stability, siRNA accumulation at the target site after administration is extremely low and requires an efficient intracellular vehicle5. Cationic liposomes emerged as safe nucleic acids carriers with the potential to transfer large pieces of DNA/RNA into cells by encapsulating and protecting the nucleic acids from enzymatic degradation6. Also, cationic liposomes spontaneously interact with DNA/RNA, thus promoting gene transfer to the cells2<....

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1. Cationic Liposomes Preparation

  1. Autoclave a clean round-bottom flask to be used for lipososmes preparation.
  2. Prepare stock solutions:
    1. - Dissolve 200 mg of dimethyl dioctadecyl ammonium bromide (DOAB) in 10 ml chloroform using a small glass bottle.
    2. - Dissolve 200 mg of cholesterol in 10 ml chloroform using a small glass bottle.
    3. - Prepare and autoclave to sterilize 5% glucose in 100 ml of RNA/DNA-ase free distilled water.
  3. Rinse .......

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ITSN-1s protein and mRNA levels were monitored at several time points after siRNAITSN delivery by Western blot and conventional and quantitative PCR as in4. ITSN-1s protein and mRNA levels in siRNAITSN-treated mouse lungs were about 75% lower by reference to controls during continuous knockdown of ITSN-1s for 21 days. Without ITSN-1s, dynamin-2, a major interacting partner of ITSN-1s and essential player in detachment of caveolae from the plasma membrane is not efficiently recruited to th.......

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Based on previous studies published by others9 and us1, 18 we developed this methodology for long term knock down of ITSN-1s in vivo by repeated intravenous administration (every 72 hr, for 24 consecutive days) of specific siRNA/liposome complexes. This experimental approach is efficient, can be used safely and repeatedly and it can be easily extended to study the involvement of a gene encoding any protein of interest in lung endothelium and pulmonary homeostasis. Under the experimen.......

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This work was supported by National Institute of Health Grants R01HL089462 to SP.

....

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number
Dimethyl Dioctadecyl Ammonium Bromide (DOAB) Sigma-Aldrich D2779
LiposoFast stabilizer (mini-extruder) Avestin Inc. LF-STB
Polycarbonate membrane, 19 mm diameter, 50 nm pore Avestin Inc. LFM-50
Cholesterol Sigma-Aldrich C-8667
Chloroform Mallinckrodt Chemicals 4432-04
Hank's balanced salts Sigma-Aldrich H1387
Round-bottom flask Fisher Scientific FHB-275-030X
Mouse siRNAITSN - on target Dharmacon/ThermoScientific J-046912-11
Peristaltic pump Ismatec REGLO-CDF digital with RHOO pump head
Ventilator Hugo Sachs Electronik NA
Barbital Sigma-Aldrich B-0500
Uranyl acetate EM Sciences 22400
Epon812 EM Sciences Discontinued by the manufacturer
Propylene oxide EM Sciences 20400
Embedding molds EM Sciences 69923-05
Tannic acid EM Sciences 21710
8 nm gold-albumin tracer Prepared in the laboratory as in16
Comments
Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)
Pyrex, 100 ml
Modified siRNA, in vivo
Part of IDEX corp.
D-79232 March, Germany
Replaced with Embed812, cat. # 14120

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