Viral-Mediated Labeling and Transplantation of Medial Ganglionic Eminence Cells for In Vivo Studies

Harvest the medial ganglionic eminence tissue, containing cortical interneuron progenitors, from genetically modified mouse embryos. 

Add a cationic polymer and dissociate the tissue into a single-cell suspension.

Add lentiviral vectors carrying a Cre recombinase-dependent GFP reporter flanked by lox sites. Incubate.

The polymer neutralizes the viral- and host-cell-membrane charges, enabling viral fusion and RNA release.

The viral RNA is reverse-transcribed and integrated into the host genome.

Centrifuge and discard the supernatant. Absorb excess media.

Transfer the cells to a hydrophobic surface and load them into an injection device.

Inject the cells into an anesthetized mouse pup's cortex.

Allow the pup to recover and grow.

The transplanted progenitors differentiate into mature interneurons in the cortex.

A subgroup of these interneurons co-expresses Cre recombinase and RFP.

In these interneurons, Cre excises the lox sites, flipping the GFP gene and enabling its expression.

Visualize the RFP and GFP co-expression to identify Cre-expressing interneurons.

After preparing lentivirus and dissecting the MGE according to the directions in the written protocol, collect both hemispheres of the MGE and put the tissue into two 1.5-milliliter collection tubes containing 500 microliters of DMEM with 10% FBS.

Keep the samples on ice until all tissue is collected. Then transfer the tubes to a BSL-2-certified hood. Carefully aspirate the media without disturbing the MGE tissues settled at the bottom of the tube.

Then add around 500 microliters of pre-warmed DMEM with 10% FBS media. Add polybrene to a final concentration of 8 micrograms per milliliter, and titrate through a P1000 pipette tip until a single-cell suspension is achieved. Then, add 15 to 20 microliters of the concentrated lentivirus to each tube.

Next, securely close each tube, invert to mix, and then place all tubes in a 37 degrees Celsius incubator. Incubate the cells with the lentivirus for at least 30 minutes, but not longer than an hour, as longer times have resulted in decreased cell viability. Invert the tubes every 10 minutes.

Following the incubation, remove the tubes from the incubator, and centrifuge at 700 times g at 4 degrees Celsius for three minutes to pellet the cells. In the BSL-2 hood, remove the supernatant and discard into 10% bleach. Next, add 1 milliliter of DMEM with 10% FBS, and titrate the pellet two to three times to wash.

After centrifuging as before, repeat this wash step two to three more times to remove excess virus. After the final wash, remove as much media as possible, and put each tube on ice.

Due to residual media on the sides of the tube, approximately 2 to 3 microliters of media will end up covering the cell pellet, by the time the transplantation procedure has begun. While ensuring that sterile technique is used throughout, draw up mineral oil into a 1-milliliter syringe, and then with a 30.5 gauge needle, back-fill the glass pipette completely with the mineral oil.

Next, attach the glass pipette to the stereotaxic device and load onto the plunger. With an assistant using the hydraulic drive, move the plunger about halfway into the glass pipette, removing the mineral oil that spills out with a clean paper towel.

Next, twist the corner of a sterile paper towel into a fine point, and use this to delicately remove excess media above the MGE cell pellet. This will concentrate the cell density so that smaller injection volumes can be achieved.

Next, use a P2 pipette to slowly draw up the cell pellet and titrate one to two times before dispensing onto a hydrophobic surface. The volume should be just under 1 microliter.

Move the tip of the needle into the cell suspension, and using the hydraulic drive, draw up the cell suspension into the pipette. After anesthetizing a pup via hypothermia, check for the effectiveness of anesthesia by pinching the skin between the toes with forceps. No reaction should be seen.

Next, place the pup onto a mold that is under the injection device, and make the skin taut by pulling back the skin on the head and then securing with standard lab tape. Working quickly, manipulate the stereotaxic arm to position the tip of the glass pipette on the pup's head, perpendicular to the surface of the head.

When the pipette is in contact with the head, consider this a zero. Next, push the pipette through the surface of the skin and skull into the cortex. Then, insert and retract the pipette two times before stopping.

For targeting of cells into the neocortex, injections are performed when the micropipette is at a depth of 0.1 millimeter. Once the needle is in position, rotate the hydraulic device to advance the plunger into the glass pipette, and inject 50 to 70 nanoliters per site.

Repeat this procedure at three to six different sites at different rostral-caudal levels if a wide distribution of transformed cells throughout the neocortex is required. When injections are complete, remove the pup from the stage and mark it according to approved procedures.

Once the pup has recovered and is able to move on its own, put it back with the lactating female and the rest of the litter. Check the pups after the procedure and the following day, to ensure that there are no signs of deteriorating health.