Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

The overall goal of this procedure is to quickly virally transduce GABAergic into neuron progenitors MGE cells before transplantation into a host for in vivo studies. This is accomplished by first isolating MGE cells from a Cree positive donor embryo. The second step is to quickly and efficiently label isolated MGE cells with a virus engineered for a specific purpose, such as labeling a specific cell type.

Next, the trans use cells are transplanted into a host and allowed to develop in vivo. Ultimately, scent microscopy or other techniques can be used to assess the transduced and transplanted cells at the appropriate time. One of the main advantages of this approach over other techniques like direct injection of virus, is that MGE cells migrate a long distance away from the injection site, which is a site of injury, making it possible to study the cells in a more normal environment.

Since MGE cells are progenitors of cortical and neurons a cell type involved in neuropsychiatric disorders including autism, schizophrenia, and epilepsy, we think that the techniques described here will actually help get at the biological underpinnings of some of these neuropsychiatric diseases. In addition, this technique provides a rapid manner to genetically modify these cells for in vivo studies. 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 two certified hood. Carefully aspirate the media without disturbing the MGE tissue settled at the bottom of the tube. Then add around 500 microliters of prewarm DMEM with 10%FBS media.

Add poly brain to a final concentration of eight micrograms per milliliter and triturate through a P 1000 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 degree 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 four degrees Celsius for three minutes. To pellet the cells in the BSL two hood, remove the sate and discard into 10%bleach. Next, add one milliliter of DMEM with 10%FBS and retro.

Rate the pellet two to three times to wash after centrifusion. 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 two to three 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 one milliliter syringe and then with a 30 and a half 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 P two pipette to slowly draw up the cell pellet and tri rate one to two times before dispensing onto a hydrophobic surface. The volume should be just under one 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 apart by a hypothermia check for the effectiveness of anesthesia. By pinching the skin between the toes with forceps, no reaction should be seen. Next, place the pop onto a mold that is under the injection device and make the skin tart by pulling back the skin on the head and then securing.

With standard lab tape working quickly manipulate the stereotypes 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 the targeting of cells into the neocortex. Injections are performed when the micro pipette is at a 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 cordal 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 in this experiment. E 13.5 somatostatin iris crew positive AI 14 flock plus MGE cells rather transplanted or transduced with the CAG flex GFP lentivirus before transplantation into wild type neo cortices and assessed at seven days post transplantation. These immunofluorescent images of neo cortices at seven or 35 days post transplantation show representative transplanted tomato positive MGE transplanted cells here immunofluorescent images of MGE cells transfuse the CAG flex GFP lentivirus and assessed at seven DPT are shown.

This histogram shows the quantification of the proportion of 2D tomato positive cells that expressed GFP at seven days post transplantation. For the following images, MGE cells were harvested from E 13.5 somatostatin IRIS CRE positive mice and transduced with a CAG flex GFP lentivirus and transplanted into the neocortex of P one wild type hosts before assessment. At 35 days post-transplant, this representative image from the neocortex of transplanted hosts demonstrates expression of GFP from the Flex Vector.

Here the tissue has been stained for somatostatin to determine co-expression of GFP and somatostatin expressing GABAergic neurons, and here the tissue has been stained for par albumin. These merged images cos stained with DAPI demonstrate the extent of colocalization of GFP with somatostatin and par albumin. Finally, this histogram shows quantification of the proportion of GFP cells that co-expressed somatostatin or par albumin Following the methods in this procedure, other molecular tools, for example, a dental associated virus may also be used to mge cells for in vivo Studies Don't forget that working with lentiviruses can be extremely hazardous work precautions, such as proper PPE and working in A BSL should always be taken while performing this procedure.