The overall goal of this procedure is to enable researchers to design and package viruses that express a fluorescent protein, CAS9 and sgRNAs, then inject them stereotactically into the mouse brain. This method can help answer specific questions in the neuroscience field such as the role of genes that underlie neurological and neuro-developmental disorders. The main advantage of this technique is the ability to manipulate specific genes without the time and resource intensive process of creating genetically engineered animals.
Prior to readying the cells, use a website to generate a 20 nucleotide guide strand or sgRNA which will be used to design single stranded oligos. After ordering oligos and using them to create a final plasmid as described in the text protocol, continue by pipetting all of the th-od cells from a cryotube into a 15 milliliter conical tube. Next add 2 milliliters of pre-warmed CO2 equilibrated complete IMDM.
Then centrifuge the cells for five minutes at 500 times G.Afterward, aspirate the supernatant and resuspend the cell pellet in 10 milliliters of complete IMDM. Plate the cells on to a 10 cm cell culture dish and incubate them overnight at 37 degree celsius in a 5%carbon dioxide incubator. 24 hours after plating, change the media by aspirating the existing media and adding 10 milliliters of pre-warmed IMDM.
Split the cells once they are confluent. To split the cells, aspirate the media and wash the plate with 5 milliliters of PBS. Then add one milliliter of 0.25%trypsin to the plate and incubate at 37 degrees celsius until the cells lift off the plate.
Afterward, add 0.5 milliliters of IMDM to neutralize the trypsin reaction and pipette the cells into a 1.5 milliliter tube. Next, spin the cells at 500 times G for five minutes. Resuspend the cells in one milliliter of IMDM and dilute 10 microliters of cells in 90 microliters of PBS.
Count the cells using either a haemocytometer or an automated cell counter and replate the cells with complete IMDM. On day five, change the media two hours prior to transfection and ensure that there is exactly 10 milliliters of media in the 10 centimeter plate. Next, prepare the transfection reagents for two disshes using two five milliliter round bottom polystyrene tubes.
Label the first tube as DNA and the second tube as 2X HBS. Then adjust the DNA concentration to one microgram per microliter in Tris EDTA at PH 7.4. To make transfection reagents for Lentiviruses and retroviruses, slowly add the required components to the tube labeled DNA while continuously tapping on the tube to mix.
After that, add one milliliter of 2X HBS to the tube labeled 2X HBS. Next, slowly add one milliliter of the contents from the DNA tube to the 2X HBS tube, one drop at a time. Continuously tap the 2X HBS tube and observe the calcium phosphate precipitate formed after each drop to ensure successful creation of the transfection complexes.
Subsequently incubate the tube in the dark for 30 minutes at room temperature. After 30 minutes, add one milliliter of the transfection reagent in slow droplets to each 10 centimeter cell plate and incubate the cells overnight at 37 degree Celsius. Replace the media on day six.
On day seven, transfer the media containing viral particles with a 10 milliliter serological pipette to a 50 milliliter conical tube before storing it at four degree celsius. Then add eight milliliters of the 0.5%FBS media to each cell plate. On day eight, collect the cell media containing viral particles and combine it with the previous day's harvest in the 50 milliliter conical tube.
In this step, centrifuge the viral supernatant at 2000 times G for 10 minutes. Afterward purify the viral media by filtering it through a 0.45 micrometer low protein binding syringe filter. Next, add 5X polyethylene glycol 6000 solution to the media.
Invert the tube several times to mix. Then incubate the viral mixture at four degree celsius for at least 12 hours. On day nine, centrifuge the viral mixture at 2500 times G for 45 minutes.
After 45 minutes, discard the supernatant and spin it again for two minutes. Followed by the removal of the supernatant again. Afterward, resuspend the pellet by adding 320 microliters of sterile PBS and incubate on the shaker at room temperature for 30 minutes.
The next day, aliquot the virus and freeze the aliquots at negative 80 degree celsius. To begin this procedure, shave the head of an anesthetized mouse in order to prepare for the injection site. Direct 4%isoflurane into the nose cone.
Next place the mouse in the stereotactic instrument. Insert the bite bar into its mouth until its teeth drop into the slot before securing the ear bars. Ensure that the animal's body is on the heating pad and the nose is situated in the nose cone.
Next, confirm the anesthesia by giving a foot pinch. Then apply artificial tears to lubricate the eyes. Subsequently swab the shaved head with povidone iodine and lidocaine.
Now make a small incision along the scalp mid-line. Dry the skull with a swab and apply hydrogen peroxide to help visualize the bregma. Using a dissecting scope, locate the bregma on the skull and place the drillbit above it.
Set the digital stereotactic coordinates of the X, Y, and Z planes to zero. In order to ensure that the head is leveled on the rostral caudal Y axis, place the drillbit on lambda and level the head so that the Z coordinate is roughly equal to zero at bregma and lambda. To ensure that the head is leveled along the X axis, place the drillbit on the midpoint between bregma and lambda and measure the Z coordinates at one millimeter from the midpoint on both sides to ensure that they are equal.
Next lower the isoflurane to 2%before placing the drill over the desired coordinates and drill through the skull carefully. After drilling all the holes, affix the virus filled syringe on to the stereotactic instrument. Center the syringe over the hole and set the Z coordinate at the skull to zero.
Slowly lower the syringe to the deepest Z depth and begin injection at a rate of 0.25 microliters per minute using a stereotactic injector. After the injection is complete at the deepest Z depth, wait one minute before raising it to the next coordinate and begin injection again. Continue this pattern until all the Z injection coordinates are injected.
After the last injection, wait two minutes before removing the syringe. Then remove the mouse from the stereotactic instrument and suture the scalp. Apply lidocaine and anti-bacterial cream to the surgical site and administer pain medication before transferring the animal to a heated recovery chamber.
This 10X wide field fluorescent image demonstrated that the high titer retroviruses infect a large number of cells whose morphology can be assessed via fluorophore expression. In this example, viruses expressing GFP or mCherry were coinjected into the dentate gyrus. Using a system of retroviruses, one can use one virus to make one genetic manipulation marked by GFP and another manipulation marked by mCHerry and then assess the single or additive changes due to each virus.
This is a 3D reconstruction of the injection extent in which closed contours of the viral spread were traced over 21 serial sections. The contour tracings were then aligned to generate 3D images for volume quantification. Total viral spread is shown in green and dendate localized spread is shown in purple.
This image shows the virus spreading along the needle track and the corpus callosum in addition to filling the rostral caudal axis of the dentate gyrus. Following the in vivo viral injection, we use other methods such as histology, electrophysiology, or live twofold on microscopy to study the consequence of the genetic manipulation on the neuronal development.
