The overall goal of the following experiment is to assess the neurological and cognitive effects of micro RNA overexpression in mice. This is achieved by preparing micro RNA expressing lentivirus as a second step. The lentivirus is injected into a specific brain region, which allows the micro RNA to overexpress and perform regulation upon its targets.
Results are obtained that show cognitive effects of micro RNA overexpression due to the anatomically targeted delivery of the lentivirus into the mouse brain. The main advantage of this technique over existing methods like genetic engineering, is that genetic engineering is a much more laborious and time consuming technique than direct lentivirus injection. In addition, lentivirus injection enables complete spatial and temporal control and follow up of the molecule being over expressed.
This method can help answer key questions in the neurono mirror field, such as what would be the effect of an over or under expression of a specific mirror on learning and memory as assessed by behavioral tests, The implications of this technique extend toward therapy of neurodegenerative diseases because microRNAs are amenable for therapeutic interventions. Incubate plated packaging cells until they reach 90%confluence, then transfect them first, change their medium to serum free. DMEM supplemented with one millimolar glutamine and 50 milligrams per milliliter.
Penicillin streptomycin then cot transfect the cells with A-P-L-K-O 0.1 pur vector plasmid coding for the delta R 8.2 and VSVG moieties and plasmid with the micro RNA of interest. Use 10 microliters of one milligram per mil, polyethaline as a carrier at 24 hours and 48 hours. Post transfection, collect the packaged lentiviruses and filter them through a 0.45 micrometer filter.
Concentrate the filtrate with lentiviruses in an ultracentrifugation at 15 degrees Celsius and 70, 000 Gs for two hours. Discard the supernatant and we suspend the lentivirus in just enough deionized distilled water required to fully dissolve the pellet so that there are no visible clumps. Then make aliquots of the concentrate to store at minus 70 degrees Celsius.
Next, measure the virus tighter infect packaging cells with serially diluted virus. In a 12 well plate from a milliliter of vector to a millionth of a milliliter of vector per well. The resulting titer should be at least 1 billion infectious particles per milliliter.
Weigh the animal and anesthetize it with an IP injection of a ketamine mixture. Use the volume proportional to the animal's weight as specified for each drug. Wait until the animal is fully anesthetized.
Then check for a lack of withdrawal reflex in the hind limb when there is no reflex. Inject the animal subcutaneously with the pain reliever IL as specified on the box. Place the animal under a heating lamp or on a heating pad and moisten its eyes with ointment.
Lastly, shave the area between the two ears with a trimming machine and sanitize the skin with 70%ethanol. Place the animal inside the stereo T and adjust the rods into the crevices, just interior to the animal's ears. Then use a scalpel to make an anterior posterior incision of about 1.5 centimeters between the ears and keep the incision open with surgery clamps.
Next, clean the surface of the skull with a cotton swab until the intersection between the coronal suture and the sagittal sutures is visible. This includes the bgma and the intersection between the coronal and lambdoid sutures or the lambda. Now position the tip of the syringe held in the stereotype to the bgma point at all three axes right down the coordinates.
They are the zero points. Then lift the syringe in the vertical axis so that a planer movement would not scratch the skull from the bgma. Move the syringe to the required location to hippocampal ca one, for example, down two millimeters in the AP axis, up and down 1.8 millimeters in the LM axis and down 1.5 millimeters in the DV axis.
Next, lower the tip of the syringe until it touches the skull. Mark the spot, then back the syringe away to make space for the drill at the spot. Using a fine driller, make a shallow hole in the skull bone.
Hold the driller steady and do not drill into the soft tissue. Drilling in short pulses can help While drilling. Make sure to study one hand using the other so that drilling through the bone would not extend any deeper into the soft tissue.
Withdrawal half of a milliliter of the concentrated lentivirus solution into the syringe. Place the syringe above the hole and slowly lower it vertically until it reaches the surface of the skull. Continue to lower the syringe into the brain very slowly.
Set the digital pump to 0.02 milliliters per minute to slowly inject the volume in 25 minutes. This will prevent backflow and ensure successful spreading. Start the infusion in some syringe types.
Consider inserting the syringe an extra half of a millimeter before retreating to the original coordinates and beginning the infusion while the substance is being infused. Wet the exposed area with PBS to prevent dehydration of the tissue. When completed, wait five minutes before backing out the syringe.
This allows the material to spread into the brain instead of retreating back into the syringe's path. Then remove the syringe very slowly and watch for backflows. If a backflow is observed, a fraction of the injected material was probably lost.
Seal the wound with histo acron being careful not to let histo acron leak into the animal's eyes. Next, to prevent the animal from dehydrating, inject one milliliter of warmed saline intraperitoneal. Place the animal in a recovery cage that is on a heated pad.
Watch the animal while it recovers for an hour after four to six weeks, assess the animal's navigation memory as in the Morris water maze. The lentivirus will have infected most of the targeted cells. By this time, an injection of 0.5 microliters of lentivirus into the ca.
One region of a mouse hippocampus was made as described in the protocol. The animal was later sacrificed, and 40 micron thick slices of its brain were analyzed. For GFP expression, DAPI staining can be seen in red.
In summary, this yielded an infected sphere of about one millimeter in the rostral coddle axis and about 0.5 millimeters in the medial lateral and the anterior posterior AEs. Mice injected with a micro RNA encoding lente vector were compared with controls in the Morris water maze. After three days of training, a representative control mouse path to the submerged platform shown in green was much shorter than that of an experimental animal in red.
Overall, the experimental animals had a more difficult time learning the task and the controls Once mastered. This technique can be done in about 40 minutes per mouse. With the flow rate being the only limiting factor Following this procedure.
Other methods like systematic oligonucleotides injection can be performed in order to answer questions such as to what extent this procedure changes brain to body communication. Don't forget that working with lentiviruses can be extremely hazardous and precautions such as disposable tyvac lab coat and neutral gloves should be used. In addition, glassware and plasticware containing liquid waste must be decontaminated before disposal in a biohazard bag.
