The overall goal of the following experiment is to evaluate the effects of long-term knockdown, of intersecting one s expression on mouse lungs structure and function. This is achieved by first preparing specific INA duplex liposome complexes to target mouse lung vasculature as a second step. The INA liposome complexes are delivered by retroorbital injection, and this is repeated every 72 hours for 24 consecutive days to achieve efficient inhibition of intersecting one S'S expression.
Next lung tissue of injected mice is sampled for biochemical analyses and EM surveys in order to evaluate intersect in one s protein and mRNA levels and to perform extensive morphological analyses of the lungs. The results obtained by western blot of lung lysates with specific intersect in one antibodies and Q-R-T-P-C-R analyses show specific and efficient intersect in one s knockdown for 24 consecutive days. The main advantage of this technique over existing methodology like rymers or lentiviruses, is that lung delivery of onic liposomes is reproducible, safe, non-toxic, and highly effective in lung endothelium.
We first had the idea for this method when we decided to evaluate the effects of intersecting N down in vivo and when other methods of SRNA delivery didn't work. To prepare onic liposomes add 315 microliters of a stock solution of dimethyl diod desal ammonium bromide, or DOAB to a round bottom flask, followed by 200 microliters of cholesterol stock solution and 9.5 milliliters of chloroform mixed by swirling. Attach the flask to the roto vapor set at 37 degrees Celsius and 100 or more rotations per minute.
Open the first valve for the argon completely by turning it clockwise. Always open and close this valve.First. The second valve controls the argon pressure blowing into the flask.
Open it partially to ensure gentle pressure. Once dry ice starts to drip over the tube connecting the Argonne gas tank with the roto vapor, lower the roto vapor into the water bath until the flask touches the water without being completely submerged. Turn the speed dial up to 100 to 120 RPM When all the chloroform in the flask has evaporated, stop the machine.
Turn the roto vapor's speed, dial to zero and close the argon tank valves. Remove the flak. Add two milliliters of a 5%glucose solution to the flask to resuspend the lipids.
Using a one milliliter pipette tip, scratch the bottom of the flask thoroughly to dissolve all lipids. Incubate the obtained solution in a 42 degree Celsius water bath for one hour while slowly vortexing the flask. Sonicate the solution for at least 20 to 30 minutes with the flask held up and the bottom just barely touching the cold water in the sonicate va.
Next, heat the flask in the roto vapor's water bath for 20 minutes at 42 degrees Celsius at zero speed with the round bottom immersed in water. Filter the liposomes through a 0.47 micron syringe filter, followed by a 0.22 micron syringe filter. Using a small extruder, filter the liposomes through a 50 nanometer membrane to generate a homogeneous population of uni lamellar liposome vesicles.
Transfer the liposomes to an einor tube and place on ice. Prepare the liposomes irna ITSN complexes at a ratio of eight mols liposomes to two micrograms RNA. In this example, 50 microliters of I-A-I-T-S-N.
From a 50 micromolar stock solution is added to 100 microliters of freshly prepared liposomes using A-R-N-A-D-N-A free EOR tube. Keep the mixture on ice until injection into mice. The mice used in this study are about four to six weeks old and weigh around 25 grams.
Confirm with the toe pinch that the animal is fully anesthetized before injection. The syringes used are one milliliter tuberculin syringes with the needle attached with one hand. Hold the mouse ears and turn the mouse down on its side to expose the internal angle of the eye.
Aim medial to the PIKA semi lunars and avoid touching the eyeball. Approach the lacrimal carle of the eye with the loaded syringe at a 45 degree angle in all three axes. Slowly inject the mixture, return the mouse to its cage and allow it to recover with the injected side.
Up.The mouse is treated with SIR NA targeting the ITSN one gene every 72 hours for 24 days. To begin this procedure, verify that the mouse is fully anesthetized by a lack of the withdrawal reflex. Start with a laparotomy and excise the diaphragm.
Then perform a thoracotomy exposing the lungs and heart. Remove the thymus using a StereoZoom microscope for better accuracy. Catheterize the pulmonary artery.
Next, perform a tracheostomy and intubate the mouse using the traches stoma. Set the ventilator to a rate of 150 strokes per minute and a stroke volume of 150 microliters. As an outlet, open the left trium using a peristaltic pump set at 1.5 milliliter per minute and Hank's solution warmed to 37 degrees Celsius.
Peruse the mouse lung vasculature free of blood for five minutes, followed by perfusion with the tracer for 10 minutes. After flushing the unbound tracer, perform in situ fixation of the lungs by a 10 minute perfusion of 4%formaldehyde, 2.5%gluar aldehyde, and 1%tannic acid. Collect the lungs and remove excess tissue.
Cut the lungs into small blocks and transfer them into a labeled scintillation vial containing two milliliters of a fixative mixture. To begin processing the lung tissue for electron microscopy. First, wash the lung blocks in 0.1 molar sodium Cate buffer briefly three times.
Next, fix the specimens in 4%aldehyde 2.5%glutaraldehyde in 0.1 Pipee buffer for one hour at room temperature postfix with 1%Pilate Osmium for one hour in the hood on ice the dark after rinsing once with Callen burger buffer. Incubate specimens in Callen burger buffer for two hours to overnight at room temperature. Rinse once in 50%ethanol and then dehydrate with the graded series of ethanol.
70%ethanol for five minutes, 95%ethanol for five minutes and then twice in 100%Ethanol for 15 minutes each. After that switch to 100%propylene oxide. For two 15 minute incubations.
Remove the propylene oxide and add a mixture of 50%propylene oxide and 50%epon. Eight 12 incubate overnight rotating on a wheel at room temperature on the following morning, remove the mixture and add fresh epon. Eight 12 for at least four to five hours on the wheel.
Add selected specimens into double tapered molds that are filled halfway with fresh 100%Epon eight 12. Place them to the edges, move the molds to a 60 degree Celsius incubator and let them cure for 48 to 72 hours. Chronic inhibition of ITSN one s expression for 21 consecutive days by repeated delivery of I TSN one s siRNA liposome complexes results in significantly lower levels of I TSN one protein in mouse lungs as shown in these representative results here, lung lysates from an untreated control and from treated mice were blotted with antibodies against I TSN one and actin at several time points posts irna delivery as indicated.
Demetric analysis of representative high blot CL films and quantitative R-T-P-C-R of I TSN one s mRNA Further confirm that treatment with I TSN one s siRNA liposome complexes efficiently down regulates I TSN one s protein in mouse lungs. The decrease in intersect in one levels leads to deficient endocytosis and T endothelial transport disruption of the inter endothelial barrier and pulmonary edema. These representative electron micrographs of lung tissue show open inter endothelial junctions or IJs labeled throughout their length by eight nanometer gold albumin particles.
Panel A one is a magnification of panel A, the arrowheads point to three to four gold albumin particles located close to each other in the same plan. In indicative of the wide opening of the IEJ. Gold particles are also associated with the abluminal exit of IJs indicated by the arrows in panels A one and B.Note also the limited number of CVE A and dilation of the peric capillary space indicated by the asterisk in panel, a acute downregulation of intersect in one expression, upregulated alternative transport pathways to compensate for deficient endocytosis.
These EM images show membranous rings in panels A and A one pleomorphic tubules in panel B and enlarged endosomes fused with typical cve LA in panel C, actively involved in the uptake and transport of gold albumin. A severe dilation of the perivascular space and proteinaceous edema are also observed. Prolonged inhibition of intersect in one expression for 24 days reduce the pulmonary edema in mice by partially restoring the inter endothelial barrier integrity.
EM morphological studies showed that the eight nanometer gold albumin tracer could not penetrate the IEJ at this time point, and instead formed filtration residues in the luminal introit of the junction indicated by the arrowhead. However, the perivascular spaces or PVS showed some dilation and mild edema suggesting that junctions are impermeable to this size of the particles, but still leaky multivesicular bodies or MVB in close proximity to the plasma membrane have some of their internal small vesicles labeled by eight nanometer gold albumin particles. This table shows the number of endocytic trans cyto structures in control and deficient mouse lung endothelial.
A significant finding is the decrease in calvi, A number in ITSN one deficient mouse lung endothelium. Chronic inhibition of intersecting one s expression caused the activation of alternative endocytic cyto pathways and partially cavi a number, the number of membranous ring or tubules labeled by gold albumin particles. In I tsn one chronic deficient mouse lung endothelium also increased by 14 fold when compared to controls.
Following this procedure, other methods like Eliza applied a mouse lung lysates can be performed in order to quantitatively estimate the trans endothelial transport of different hnic tracers. After watching this video, you should have a good understanding of how to specifically deliver complexes of onic liposome, SRNA to mouse lung endothelium in order to knock down the protein of interest and to evaluate its function.
