The overall goal of this procedure is to manipulate gene function during the postnatal period of brain development. This is accomplished by first selecting and preparing the appropriate viruses, loading them into the syringe and preparing the injection equipment. Then virus is injected into the brains of newborn mouse pups, and the final step is to sacrifice the animals and image the injected regions.
Ultimately, results can be obtained that show differential labeling of control and knockout cells through immunofluorescence microscopy. This method can help answer key questions in the neuroscience field, such as how the period of postnatal development is genetically regulated. For injection, a commercially available adeno-associated virus solution can be used at full titer, typically 10 to the 12th, genome copies per milliliter to manipulate a large number of cells.
If sparse or labeling is desired, begin with a one to 10 dilution. After thawing the virus on ice, immediately transfer the pre dilution volume to a sterile 250 microliter PCR tube. Keep the dilution on ice.Now.
Prepare the injection pump by selecting an appropriate syringe and connecting it to a loading needle. Next, gently draw up the virus solution until a very small air bubble is seen in the syringe and inert loading dye may be used to make this step easier. Now secure the syringe to the pump with a retainer.
Then gently attach connective tubing between the syringe and the needle holder. Be sure the injection needle is in direct contact with the connective tubing inside of the needle holder. Now, slowly move the solution through the connective tubing until a tiny drop of solution is visible at the needle's tip.
Carefully secure the plunger next to the pusher block with a bracket on the pump. Set the injection rate to eight microliters per minute and set the injection volume to between one and two microliters. Then adjust the pumps syringe diameter setting to match the diameter of the syringe you are using.
Lastly, to protect the pups during the recovery phase, set the hot plate to 38 degrees Celsius and cover it with a paper towel. Using an IACUC approved protocol, prepare P zero or P one mouse pups for injection by subjecting them to cold anesthesia. Dampen a few paper towels with water and place them on ice.
Then place four or five pups on the paper towel well separated. Fold the paper towels over the pups gently place a small amount of crushed ice on top of the paper towels and incubate the pups for about five minutes. The pups can be safely kept on ice for up to 15 minutes and still have a fine recovery.
Now position an anesthetized pup on a mounting block for best access to the injection site. The cortex, for example, is most easily accessed by laying the animal flat on its belly. The pup may be secured in place using a bandaid, manually stabilize the pup's head and pull the skin tight to keep it from moving.
Then navigate anatomical landmarks of the skull like the lambda suture, and select a reproducible injection point with the other hand, gently insert the needle through the skull. Do not push hard if the needle does not easily penetrate the skull. Instead, reposition the needle and gently try again.
Injection depth varies slightly between individual animals and strains for the cortex, one half to one millimeter depth usually works. Now, inject the virus solution by having a colleague activate the pump to minimize hand movements. Ideally, a foot pedal could be used to start the pump after the injection.
Keep the needle in place for a few seconds, then keeping the pup's head still smoothly. Slide the needle out. Now, allow the pup to recover for five to 10 minutes on the covered hot plate.
During this time, continue injecting other pups. When the pups have all regained their pink color and are all moving, gently rub them with a portion of their home bedding. Only then can they be returned to their mother.
Otherwise, they could be treated as unfamiliar pups, which the mother would most likely kill after the pups have been returned to their mother. Clean the injection setup by fully loading the syringe with acetone or 70%ethanol. If acetone sensitive components such as sano, acurate, adhesives are in use, flush the solution through the injection setup several times using fresh solution each time.
This will remove any remaining virus solution and precipitates the solvents will evaporate. After flushing out the setup, disinfect the working area of the bleach to leave it ready for the next experimenter.Mentor. Successful technique produces a robust neuronal infection at the injection site.
Injections into specific regions such as the cortex and superior colliculus typically generate local infections with minimal spread to adjacent regions. Using a high titer virus solution makes infection of adjacent areas such as the hippocampus, more likely injection with a low titer virus solution. Results in sparse infection typically near the injection site like the cerebellar injection at the midline, the number of cells infected should correlate with a titer of the injected viral solution.
For example, injecting a mix of high titer solutions of two R AAV eight viruses expressing different fluorescent proteins into the cerebellum, infects many kinji cells that are differentially labeled. Conversely, injecting a low titer virus solution can result in sparse infection to aid in visualizing single cells. Fluorescently labeled neurons can be observed directly in fresh cut live tissue, or they can be subjected to immuno staining.
This enhances the endogenous fluorescent signals for later image acquisition by widefield or confocal fluorescent microscopy. Lastly, RAAV eight is capable of infecting a variety of neuron types in the cortex with strong labeling of fine processes Once mastered, this technique can be done in approximately 30 minutes if it is performed properly.
