嗅神経細胞のレンチウイルスを介した遺伝子操作と可視化 in vivoで

One of the challenges of studying development in neuroscience is capturing dendritic and axonal morphologies at a single cell level in vivo. In the olfactory system, olfactory sensory neurons extend their axons from the olfactory epithelium in the nose to the olfactory bulb in the brain. As multiple olfactory sensory neurons project toward the olfactory bulb, they fasci into bundles.

This complicates the study of individual axon trajectories and their terminal morphologies. We have developed a method to visualize single olfactory sensory neurons in vivo by micro injecting A GFP construct. Bearing lentivirus.

Successful transduction with lentivirus illuminates the entire olfactory sensory axon length, including the terminal arbor. In addition to visualizing single olfactory sensory neurons, this method allows us to manipulate gene expression of a particular gene of interest to study its function in an in vivo context. This lentivirus procedure is biosafety level two and needs to be performed in a laminar flow hood.

To maintain a sterile environment, spray everything inserted into the hood with 70%ethanol in the hood, prepare and preheat a 37 degree waterbed at least 20 minutes. Before injection. Seal a plastic storage bag half full with water and set it on an empty heating block incubator.

This will allow mice to recover following injection. Fill a biohazard waste container with 10%bleach to decontaminate all virus exposed material. From this procedure, prepare a small open dish of 70%ethanol and a second dish with sterile distilled water.

In a laminar flow hood, we'll be using a five microliter capacity Hamilton syringe with a 33 gauge needle. This particular model allows you to adjust the plunger resistance by turning a tension dial located at the top of the plunger chamber. This feature provides microinjection volume control to sterilize the needle and syringe chamber.

Repeatedly aspirate and eject ethanol from the dish several times. Rinse the syringe cartridge by aspirating and ejecting sterile water several times. Place syringe safely aside in the flow hood.

Arrange alcohol wipes near the injection station. Prepare an injection stage that provides a raised surface that will ease positioning of the mouse. One can use an inverted 10 centimeter culture dish covered with Kim wipes.

Mouse pups can be anesthetized by hypothermia up until weaning age. Cut out a finger of a rubber glove and place the mouse pup in the finger cutting. Then place on ice for five minutes.

The glove will prevent skin burns that would occur from direct skin contact with the ice test for thorough anesthesia by pinching the pup's hind and four feet to make sure the animal does not respond. We are now ready for microinjection of lentivirus. We'll be demonstrating with a neonatal pup.

If an older age of mouse is used, one should substitute the 33 gauge needle for a thicker gauge needle in order to penetrate the skull's surface. Sterilize the mouse's nose surface by wiping it with an alcohol pad. Remove lentivirus stocks from freezer storage and thaw on ice.

We have produced lentivirus in the lab ourselves using established methods. In developing this method, we have found that a minimal viral titer of 10 to the ninth infectious units per mil is required for a good transduction rate. Aspirate two microliters of lentivirus stock solution to the sterilized Hamilton syringe.

Place the mouse pup on top of the injection stage. Grasp the skin on top of the head at the ears, and gently pull back. We have outfitted our flow hood with a dissecting scope.

In order to accurately direct micro injections, search the dorsal skull surface at the forebrain for an outline of the olfactory bulb. Our micro injection targets closely surround the olfactory bulb, but should not include the bulb itself. We give four injections into the olfactory epithelium of each pup.

These positions represented with blue dots require the needle to be inserted at an angle directed medially. The additional two injections represented with green dots are staggered, more medially and anterior to the olfactory bulb. These injections can be angled coddly.

The needle angles suggestions are made in consideration of the ventral positioning of the olfactory epithelium, which is mostly situated beneath the olfactory bulb. Perpendicular access is thus restricted. Insert the needle through the top of the nasal cavity about three millimeters below the skin surface.

To reach the olfactory epithelium. A trained hand should feel the drop in resistance when the nasal cavity luminal space is reached. This area should be avoided.

Micro inject 0.5 microliters of virus stock solution per position. Again, choosing two sites per side, both left and right, four injections total of 0.5 microliters. Each per mouse means two microliters of virus needed per mouse in a single injection session.

Again, be careful not to inject the olfactory bulb blot dry any residual virus solution or blood that may escape from the injection site or nostril openings. Using an ethanol wipe, dispose of wipes in the diluted liquid bleached container, immediately placed the injected mouse onto the waterbed incubator for recovery. Decontaminate needle syringe by repeating sterilization procedure performed during preparation Injected mice can be returned to their cage 30 minutes after recovery on the waterbed incubator.

This entire microinjection can be repeated again in four hours. If you'd like to increase the number of olfactory sensory neurons that will be infected, infected olfactory sensory neurons may be visualized throughout the length of the cell in as few as three days. The animals sacrificed and perfused for observation at the desired time point.

Using standard methods In order to visualize transduced single cell olfactory sensory neuron trajectories and axon termini, we dissect and section the olfactory bulb coronal at a 60 micron thickness. The section will thus contain all cell layers of the olfactory bulb, including olfactory sensory neuron axon bundles, and axon terminals, which synapse in glomerular structures bound at the bulb surface in preparation for floating immunohistochemistry. Serial sections of the olfactory bulb are made and collectively swept into a 10 centimeter culture dish with five milliliters of phosphate buffered saline.

Transfer the floating sections into a 50 milliliter conical vial. Once sections have settled to the bottom of the vial, vacuum off phosphate buffered saline. Take caution not to vacuum up.

Your sections replenish with 10 milliliters of fresh buffer to wash away any residual fixative or embedding material. Immediately pipette this resuspension into a 15 milliliter conical vial, making sure to capture all floating sections, vacuum off rinse solution, and perforate the sections with a solution of 0.3%Triton X 100 in phosphate buffer saline place on a rocker platform for 10 minutes. At room temperature, you may then proceed through a standard immuno staining protocol in the same conical vial, repeating the vacuum and rocker steps as needed for this size vessel, retain a 10 milliliter volume for staining and rinsing steps.

Here is a list of antibodies we use to cos stain positively transduced olfactory sensory neurons, and the general olfactory sensory neuron population. For thorough antibody penetration of the 60 micron bulb sections, it is recommended to incubate overnight at four degrees Celsius. Wash depth should also be no shorter than 20 minutes each.

Gently spread each section on a glass slide with a paintbrush mount each slide with flora Mount G supplemented with chromatin stain solution. Make efforts to eliminate all bubbles from the mounting medium. Mount your Sections in rows to simplify the subsequent screening step.

Scan these layers on your immuno stain bulb sections for the GFP positive axon fibers and termini. Using a fluorescent microscope positively infected nerve terminals can then be imaged by confocal microscopy. It is helpful to calibrate the focal plane in your area of interest using the DPI and olfactory marker protein channels.

These results show a single olfactory sensor neuron axon fiber and axon terminal within the glomerulus, which is CO with olfactory marker protein in red and vlu two in gray infected olfactory sensor neuron cell bodies and their dendritic knob can also be visualized within the olfactory epithelium if this tissue was included in the dissection and immunostaining steps. Hi, my confocal projection provides us with a greatly detailed morphology of a single olfactory sensory neuron axonal extension path and terminal arbor. One can see the hooking of the axon fiber back towards its glomerular target, as well as the details of its branching pattern within the glomerular neuro pill.

The lentiviral microinjection method allows visualization of single cell axon, morphologies of olfactory sensory neurons, and allows studies that manipulate gene expression, lentivirus transduction results in stable genomic integration of constructs. Lentivirus does not trans synaptically infect neighboring cells. Visualization can be achieved in as few as three days and has been confirmed to last at least three months and likely persists for longer periods.

Olfactory sensory neurons are not readily infected by nasal infusion of lentivirus solution. Therefore, microinjection through the surface is required in this procedure. So that's our lentivirus Microinjection procedure.

I hope you've found all this information useful towards achieving single cell morphologies of your own Happy hunting.