The overall goal of this adeno-associated virus vector gene transfer is to genetically manipulate the rat's brain and spinal cord on an expansive wide scale basis. This method can help answer key questions in the gene transfer field. It is a very efficient way to introduce genes in-vivo and therefore, a very powerful, a very versatile technique.
This procedure is both rapid and highly reproducible. We have used this wide scale gene transfer approach to express an amyotrophic lateral sclerosis related protein throughout the spinal cord of rats in several publications. My colleague, Robert Dayton will be demonstrating the procedure.
Mychal Grames will be assisting Robert. Begin by preparing the workstation, first, clean the surface with 70%ethanol. Then place a heating pad in the work area and set it to warm to 37 degree celsius.
Place a bench pad on top of the heating pad. Check that there is adequate oxygen and isoflurane for the procedure. Clean the induction chamber and anesthesia mask with 70%ethanol and place the anesthesia mask on the bench pad.
Make sure that the following are in hand, one paraffin film square of about 5 x 5 centimeters per animal, one alcohol preparation pad and two or three gauze pads per animal, pipette tips and a micropipette. Then, thaw the vector at room temperature and label one sterile microcentrifuge tube for each animal. Pipette the required volume of AAV into the appropriate tube and bring the volume up to the desired injection volume with lactated ringer solution or saline.
Post vortex the sample for one or two seconds and then, pulse centrifuge for five seconds to bring the sample to the bottom of the tube. Pipette the total injection volume out of the tube and onto the square of paraffin film. Next, place a 30 gauge needle on a one milliliter syringe.
Place the needle with the bevel facing down into the vector on the paraffin film. Pull back on the syringe plunger until all the virus is in the needle and syringe. Properly anesthetize the rat using the anesthesia system.
Ensure an adequate plane of anesthesia by the absence of reaction to a toe pinch. Then, place the rat in the work area on its side and maintain anesthesia using the nose cone. With the rat lying on its side, one lateral tail vein should be facing up.
The injection is two thirds down the length of the tail. Wipe the injection site with the alcohol preparation pad. Firmly hold the tail slightly above the injection area, with one finger directly over the lateral tail vein, this will dilate the vein.
With the bevel facing up, align the needle with the visible tail vein in the same direction. Pierce the skin over the tail vein, while taking great care to avoid the other hand holding the tail and pressing on the tail vein. Release pressure from the tail vein to allow for normal blood flow.
Then move the needle into the vein. To ensure that the needle has punctured the tail vein, pull back slightly on the plunger. If the needle is in the vein, blood will flow into the needle.
After repositioning the needle if required, stabilize the needle by holding the end of the syringe against the tail. Slowly inject the vector into the tail at approximately 20 microliters per second. In the case of a poor injection, the animal will only be partially transduced and the results will probably not be useful.
However, with practice, good injections are very reproducible and reliable. If the vector has been administered, remove the needle from the tail and press a gauze pad against the injection site to help stem the bleeding for 30 to 60 seconds. Turn off the isoflurane anesthesia and oxygen.
Monitor the rat until it regains consciousness and then return it to its cage. Dispose the materials that maybe contaminated with AAV into an appropriate biohazard container and clean the workstation with 70%ethanol. Obtain 50 micron coronal sections from the fixed and cryo-protected brain of an AAV injected animal, using a sliding microtome with freezing stage.
After immunostaining for GFP and mounting on slides, photomicrograph each section in the defined region of interest with a low magnification lens using a microscope. Keep the camera settings the same across all samples to be analyzed and save the data as TIFF files. Open the photomicrograph in the widely available Scion Image program.
Two windows will open, close the window indicated as index color. Choose options, then density slice. A range of shades will be indicated in red on the look up table window.
In the window, use the cursor to fill in the specific GFP labeling only and stop when the non-specific background begins to be picked up in the image. Next, click analyze and set scale. The fourth line should say units, from the drop down list, choose pixels then click okay.
To measure the fluorescent area, click analyze and then, measure. Then, click analyze and show results. A results window will appear and the fluorescent area measurement will be under the heading labeled, area.
Once all measurements have been recorded, average the values from three to six sections per animal. Use an appropriate statistical test to compare the transduced areas between the groups. This representative image shows GFP immunofluorescence in the rat cerebellum, four weeks after a tail vein injection of AAV 9 GFP.
Image analysis software was used to selectively highlight the fluorescent area and to quantify the highlighted pixels. This is the GFP image from a non-transduced animal. Analysis of this image, using the same parameters for the GFP injected animal, does detect a very low level of background signal.
Once mastered, the injection can be done in about five minutes per rat. In conclusion, this technique describes a way to efficiently introduce genes to the rat CNS, a one time gene delivery produces lasting expression and long term effects. Most importantly, the central nervous system is manipulated from the periphery.
A relatively non-invasive intravenous vector injection, reaches the brain and spinal cord, the CNS.
