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

1. Cationic Liposomes Preparation
<ol>
 <li>Autoclave a clean round-bottom flask to be used for lipososmes preparation.</li>
 <li>Prepare stock solutions:
 <ol class="lalpha">
 <li>- Dissolve 200 mg of dimethyl dioctadecyl ammonium bromide (DOAB) in 10 ml chloroform using a small glass bottle.</li>
 <li>- Dissolve 200 mg of cholesterol in 10 ml chloroform using a small glass bottle.</li>
 <li>- Prepare and autoclave to sterilize 5% glucose in 100 ml of RNA/DNA-ase free distilled water.</li>
 </ol>
 </li>
 <li>Rinse the round-bottom flask with chloroform. Add 5-10 ml to the flask, swirl, and let it sit for few minutes. Keep the flask covered with aluminum foil at all times. Empty the chloroform from the flask.</li>
 <li>Preparation of liposomes:
 <ol class="lalpha">
 <li>- Add 315 &mu;l of DOAB stock solution to the round-bottom flask.</li>
 <li>- Add 200 &mu;l of cholesterol stock solution to the flask.</li>
 <li>- Add 9.5 ml of chloroform to the flask and mix by swirl.</li>
 </ol>
 </li>
 <li>Set the rotavapor at 37 &deg;C, 100 rotations per minute (rpm) or more (100-120 rpm). The rotavapor should be connected to a water-pump vacuum control with a suction tube properly attached.</li>
 <li>Connect the argon gas tank and the rotavapor.</li>
 <li>Attach the flask to the rotavapor and adjust the optimal settings before lowering it in the water bath. Open the first valve for the argon completely by turning it counterclockwise. Always open and close this valve first. The second valve controls the argon pressure blowing in to the flask. The pressure should be always gentle.</li>
 <li>Lower the rotavapor in the water bath, enough for the flask to touch the water without being completely submerged and turn on the speed.</li>
 <li>Wait until all the chloroform evaporates, about 10 min. Then stop the machine.</li>
 <li>Turn off the rotavapor speed and the argon tank valves. Now the flask should be removed.</li>
 <li>Add 2 ml of 5% glucose to the flask to dissolve the lipids.</li>
 <li>Scratch the bottom of the flask thoroughly using 1 ml pipette tip to dissolve all lipids. After scratching, remove any remaining lipid from the 1 ml tip into the flask, if necessary, with a second tip.</li>
 <li>Incubate the obtained solution in 42 &deg;C water bath while slowly vortexing (15 rpm) the flask.</li>
 <li>Fill the sonicator vat with cold water. Sonicate the solution for at least 20-30 min with the flask held up and the bottom just barely touching the water.</li>
 <li>Heat the flask in the rotavapor's water bath for 20 min at 42 &deg;C, at zero speed, with the round bottom immersed in water, this time.</li>
 <li>Filter the liposomes through a 0.47 micron and subsequently, 0.22 micron syringe filters.</li>
 <li>Using a small extruder, filter the liposomes through a 50 nm membrane to generate a homogenous population of unilamellar liposome vesicles.</li>
 <li>Transfer the liposomes to an Eppendorf tube and place it on ice.</li>
</ol>
2. Prepare the Liposomes: siRNA-ITSN-1 Complexes
<ol>
 <li>Resuspend the on-target mouse siRNAITSN in siRNA buffer containing 20 mM HEPES and 150mM sodium chloride, pH 7.4, to a final concentration of 2 &mu;g/&mu;l. Keep it on ice before mixing.</li>
 <li>Prepare the liposomes:siRNAITSN complexes at a ratio of 8 nmol liposomes:2 &mu;g RNA (or 400 nmol liposomes:100 &mu;g siRNAITSN). In the present study, we added 50 &mu;l of siRNAITSN from the stock solution (50 &mu;M) to 100 &mu;l of freshly prepared liposomes using a RNA/DNA-ase free Eppendorf tube. Note that the concentration of siRNAITSN is very important because a maximum of 150 &mu;l of mixture can be bilaterally and retro-orbital injected in the mouse at one time.</li>
 <li>Keep the mixture on ice while waiting to inject the mice.</li>
</ol>
3. Mouse Retro-orbital Vein Injection (Internal Angle of the Eye Socket)
All mouse experiments were approved and performed in accordance with the guidelines of Rush University Institutional Animal Care and Use Committee.
<ol>
 <li>Anesthetize the mouse by intra-peritoneal injection of a 50 mg/kg body weight ketamine and 5 mg/kg body weight xylazine (commercially available mixture). Depending on the animal weight and age, adjust the anesthetics volume and concentration. The mice used in the study mice are CD1 male mice, 6-8 weeks old and weigh around 25 grams. The syringes are tuberculin 1 ml type with the needle attached.</li>
 <li>Hold mouse ears with one hand and turn the mouse down on a side to expose the internal angle of the eye. Aim medial to the plica semilunaris; avoid touching the eyeball.</li>
 <li>Approach the lacrimal caruncle of the eye holding the syringe containing the mixture at a 45 degrees angle in all three axes.</li>
 <li>Slowly inject the mixture and let the mouse to recover with the injected side up. If a large liquid droplet forms at the needle insertion place, retract the needle, suction back the droplet into the syringe and try again. Repeated injections are best done by alternating the eyes, to allow perfect recovery. If unsure, this technique can be checked by injecting blue dye (50 &mu;l, 0.5% crystal violet in methanol) and exposing the mouse lungs to observe the blue vasculature.</li>
 <li>The mice in this study were injected every 3rd day for 3 weeks without fatalities or adverse effects.</li>
</ol>
4. Mouse Lung Perfusion and Tissue Collection for Biochemical Studies
<ol start="0">
 <li>Set the peristaltic pump to run at 1.5 ml/min.</li>
 <li>Prepare the setting: ventilator, operating table, instruments, sterile cotton swabs, q-tips, strings, beakers, distilled water, Hank's balanced salt, tracer.</li>
 <li>Anesthetize the mouse by intraperitoneal injection of the anesthetic mixture (100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine).</li>
 <li>At the neck level, remove the salivary glands and expose the trachea.</li>
 <li>Start with laparotomy, excise the diaphragm, and follow with thoracotomy, exposing the lungs and the heart.</li>
 <li>Remove the thymus.</li>
 <li>Using the light microscopy for better accuracy, catheterize the pulmonary artery.</li>
 <li>Do tracheostomy and intubate the mouse using the tracheostoma. Set the ventilator to a rate of 150 strokes/min and stroke volume of 150 &mu;l.</li>
 <li>As outlet, open the left atrium.</li>
 <li>Perfuse the mouse lung vasculature free of blood for 5 min, using the peristaltic pump and Hank's solution warmed at 37 &deg;C.</li>
 <li>Perfuse the tracer (8 nm gold-albumin16) for additional 10 min at the same flow rate.</li>
 <li>Flush the unbound tracer by 5 min lung perfusion with Hank's solution.</li>
 <li>Follow with in situ fixation of the lungs by 10 min perfusion of 4% (wt/vol)formaldehyde, 2.5% glutaraldehyde, and 1% tannic acid in 0.1 PIPES buffer, pH 7.2.</li>
 <li>Collect the lungs, remove the excessive tissue, cut small blocks (3/3 mm), and place them in to a labeled scintillation vial containing 2 ml of fixative mixture. </li>
</ol>
 
The mice will expire fully anesthetized during the procedure.
5. Mouse Lung Tissue Processing for EM
<ol>
 <li>Prepare stock solutions: 1% Palade OsO4, acetate veronal, and Kellenberger buffer.
 <ol class="lalpha">
 <li>- 1% Palade-OsO4 buffer contains: 1 ml acetate veronal stock, 1.25 ml 4% OsO4, 1 ml of 0.1 N hydrochloric acid, and distilled water up to 5 ml of final volume.</li>
 <li>- Acetate veronal stock solution is obtained by dissolving 1.15 g sodium acetate andydrous and 2.94 g barbital in 100 ml of distilled water.</li>
 <li>-Kellenberger solution contains 2 ml of acetate veronal stock, 2.8 ml of 0.1 N hydrochloric acid, 0.05 g uranyl acetate, and 5.1 ml of distilled water, pH= 6.</li>
 </ol>
 </li>
 <li>Wash the lung blocks in 0.1 M sodium cacodylate buffer, pH= 7.4, briefly 3 times.</li>
 <li>Fix the specimens in the 4% (wt/vol) formaldehyde, 2.5% glutaraldehyde in 0.1 PIPES buffer, pH 7.2, for 1 hr, at room temperature.</li>
 <li>Post-fix with 1% Palade-Osmium for 1 hr in the hood, on ice, in the dark.</li>
 <li>Rinse once with Kellenberger buffer.</li>
 <li>Incubate specimens in Kellenberger buffer for 2 hr to overnight, at room temperature.</li>
 <li>Rinse once in 50% ethanol.</li>
 <li>Dehydrate with graded series of ethanol, 5 min/each: 70%, 95%, then 2 x 15 min 100% ethanol.</li>
 <li>Switch to 100% propylene oxide, 2 x 15 min.</li>
 <li>Remove the propylene oxide and add a mixture of 50% propylene oxide and 50% Epon 812, incubate overnight, rotating on a wheel, at room temperature.</li>
 <li>Next morning, remove the mixture and add fresh Epon 812, at least for 4-5 hr on the wheel.</li>
 <li>Using doubled tapered molds, filled half way with fresh 100% Epon 812, add selected specimens and place them to the edges.</li>
 <li>Move the molds to the incubator set at 60 &deg;C and let them cure for 48-72 hr.</li>
 <li>When polymerized, send the blocks to be thin (60-70 nm thick) sectioned.</li>
</ol>

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The authors have nothing to disclose.

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...

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. More recently liposomes have been applied to deliver vaccines and low-molecular-weight drugs7. Liposomal delivery of microRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells8. When targeting the vascular endothelium, the intravenous delivery is essential because the complexes are unlikely to cross the endothelial barrier and extravasate in to the interstitium9. By comparison to other organs, ECs from the microvasculature of the lung have the greatest uptake and avidly internalize cationic liposome and DNA/RNA complexes, followed by the lymph nodes and Peyer's patches9. Intravenous delivery in rodent models of siRNA via retro-orbital or tail vein injections has been proven harmless even in high concentrations, such as 50 mg/kg10. In the published literature the composition of cationic liposomes differs, based on the lipids formulation and their equimolar ratio11, 12. There is a wide array of potential applications using cationic liposomes for gene delivery in vivo, whether targeting down-regulation/over-expression of the proteins, delivery of vaccines or anti-tumor therapies13-15. Important to remember is that the efficiency of DNA/RNA-cellular membrane interaction is regulated by the shape coupling between generated complexes and membrane lipids. This suggests that tailoring the lipids composition to the targeted cellular membrane profiles may be beneficial and result in high rates of transfection in a particular cell type6.

<table><tbody><tr><td>Dimethyl Dioctadecyl Ammonium Bromide (DOAB)</td><td>Sigma-Aldrich</td><td>D2779</td><td></td></tr><tr><td>LiposoFast stabilizer (mini-extruder)</td><td>Avestin Inc.</td><td>LF-STB</td><td></td></tr><tr><td>Polycarbonate membrane, 19 mm diameter, 50 nm pore</td><td>Avestin Inc.</td><td>LFM-50</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C-8667</td><td></td></tr><tr><td>Chloroform</td><td>Mallinckrodt Chemicals</td><td>4432-04</td><td></td></tr><tr><td>Hank&#039;s balanced salts</td><td>Sigma-Aldrich</td><td>H1387</td><td></td></tr><tr><td>Round-bottom flask</td><td>Fisher Scientific</td><td>FHB-275-030X</td><td></td></tr><tr><td>Mouse siRNA&lt;sub&gt;ITSN&lt;/sub&gt; - on target </td><td>Dharmacon/ThermoScientific</td><td>J-046912-11</td><td></td></tr><tr><td>Peristaltic pump</td><td>Ismatec</td><td>REGLO-CDF digital with RHOO pump head</td><td></td></tr><tr><td>Ventilator</td><td>Hugo Sachs Electronik</td><td>NA</td><td></td></tr><tr><td>Barbital</td><td>Sigma-Aldrich</td><td>B-0500</td><td></td></tr><tr><td>Uranyl acetate</td><td>EM Sciences</td><td>22400</td><td></td></tr><tr><td>Epon812</td><td>EM Sciences</td><td>Discontinued by the manufacturer</td><td></td></tr><tr><td>Propylene oxide</td><td>EM Sciences</td><td>20400</td><td></td></tr><tr><td>Embedding molds</td><td>EM Sciences</td><td>69923-05</td><td></td></tr><tr><td>Tannic acid</td><td>EM Sciences</td><td>21710</td><td></td></tr><tr><td>8 nm gold-albumin tracer</td><td></td><td>Prepared in the laboratory as in16</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)</td></tr><tr><td colspan="4">Pyrex, 100 ml</td></tr><tr><td colspan="4">Modified siRNA, &lt;em&gt;in vivo&lt;/em&gt;</td></tr><tr><td colspan="4">Part of IDEX corp.</td></tr><tr><td colspan="4">D-79232 March, Germany</td></tr><tr><td colspan="4">Replaced with Embed812, cat. # 14120</td></tr></tbody></table>

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

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.

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...

Watch this Scientific Journal Video about Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Elect

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

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 ...

long-term-silencing-intersectin-1s-mouse-lungs-repeated-delivery

Research

JoVE Journal

Biology

10.6K Views.  Rush University. 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.

Video: 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

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

Genetics

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Medicine

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Bioengineering

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

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

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Chapters in this video

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling