The overall goal of this procedure is to transfect primary rodent postnatal neuroblast in order to examine the effect of target proteins on their migration. This is accomplished by first dissecting neuroblast from the rostral migratory stream of the rodent pops. The second step is to dissociate them and to transfect them by nucle affection with either DNA or S-H-R-N-A.
Next, the neuroblast are re aggregated in hanging drops and then cultured in suspension for an appropriate time. The final step is to embed the re aggregated neuroblast clusters in a three dimensional matrix and leave them to migrate. Ultimately immunofluorescence or time-lapse imaging microscopy is used to analyze neuroblast migration.
This method can help answer a key question in the neurogenesis field, such as the control of stem cell derived neuroblast migration in the postnatal brain. The main advantage of this technique is that it is relatively easy and fast compared to the use of viral vectors, which can be challenging and time consuming. After sacrificing a P six to P seven rat litter, make an an posterior incision in the skin along the mid sagittal suture from the nose to the cerebellum with a scalpel blade on each rat.
Next, peel the skin off. Repeat the same incision along the skull. Then gently remove the cranial flaps with forceps and carefully remove the brain together with the olfactory bulbs with a spatula.
After that, cut the most coddle third of the brain and discard it. Subsequently, chop the brain tissue into 1.4 millimeter thick coronal slices using a tissue chopper. Transfer the slices to a dish containing cold dissection medium.
Then view the slices under the dissection microscope. Carefully separate them using a needle. The rostral migratory stream appears as a triangular translucent area in the center of OB sections and as a small circular area.
In more coddle brain slices, cut the RMS out of each slice with a microsurgical knife taking care to avoid including surrounding tissue. Next, collect the RMS fragments with a plastic paster pipette and place them in a small dish containing cold dissection medium on ice. Then gently rotate the dish to gather the RMS fragments in the center of the dish.
Collect the fragments with a plastic pipette and transfer them into a 15 milliliter tube. Leave the fragments to settle at the bottom of the tube. Replace the dissection medium with two milliliters of dissociation medium T tri.
Rate the RMS fragments by gently pipetting the fragment suspension up and down about 10 times using a P 1000 pipette afterward. Leave the tube with the tissue fragments in a 37 degree Celsius water bath for two minutes. Subsequently, pipette the solution again 10 times and ensure that the fragments have dissociated.
Then inactivate the trypsin by adding five milliliters of pre wormed DMEM plus 10%FCS and centrifuge the cell suspension at 433 times G for five minutes. Remove the excess medium and resus. Suspend the cell pellet by gently pipetting it in one milliliter of prewarm DMEM plus 10%FCS, and then add another four milliliters of the same medium.
Then perform a cell count. A minimum of 2.5 times 10 to the sixth cells are required for each nucleo affection while optimal results are achieved. Using three to four times 10 to the six cells per nucle affection.
Centrifuge the cell suspension at 433 times G for five minutes. After that, remove as much medium as possible in this procedure. Immediately Resus, suspend the cell pellet in the rat neuron nucleo affection solution previously incubated.
At room temperature transfer 100 microliters of the cell suspension to each EOR tube containing SI R-N-A-D-N-A. Then mix it gently by pipetting two to three times with a P 200 pipette. Next, add the sample to the bottom of the nucle affection vete taking care to avoid generating any bubbles.
For nucle affection use program G dash 0 1 3 for rat cells or O dash 0 0 5 for mouse cells. After that, quickly add one milliliter of pre-war DMEM plus 10%FCS to the nucle affected sample. Transfer the sample to a 15 milliliter tube containing five milliliters of pre-war DMEM plus 10%FCS using the plastic pipette provided by the nucle affection kit.
Then centrifuge the sample at 433 times G for five minutes. Carefully remove all excess medium and resuspend the pellet in 25 to 30 microliters of pre-war DMEM plus 10%FCS using a P 20 pipette. Do not use more than 30 microliters of medium.
Afterward, pipette the suspension as a drop onto the inner side of a P 35 dish lid. Then invert the lid over the P 35 dish containing two milliliters of complete medium. Leave it in the incubator at 37 degrees Celsius with 5%carbon dioxide for at least five hours.
After five hours, transfer the hanging drops from the lid into the complete medium in the dish. Using a P 1000 pipette with a cut tip. Then incubate the samples at 37 degrees Celsius with 5%carbon dioxide for 24 hours for DNA NU nuclear infections, or 48 hours for SI A HRA nucle affections.
Now prepare 25 milliliters of complete medium and pre equilibrate at 37 degrees Celsius with 5%carbon dioxide for a few hours. In the meantime, take the frozen aliquots of the basement membrane matrix out from the negative 80 degree Celsius freezer and thaw them on ice in the cold room. For each nucle affection, prepare a six centimeter dish containing up to eight 13 millimeter sterile cover slips.
Place the dishes on an icebox covered with cling film. To maintain humidity, place a strip of damp tissue inside a 15 centimeter dish that will be used to hold up to three six centimeter dishes containing knee embedded oblasts. Next, add the complete medium to the haw matrix in a one to three ratio, transfer the re aggregated cell clusters to a 15 milliliter tube and centrifuge it 433 times G for five minutes.
After that, remove the excess medium and resuspend the pallet in 10 microliters of complete medium. Then place two microliters of cell aggregate suspension onto each sterile cover slip. Add 18 microliters of matrix complete medium mixture and use the pipette tip to spread the matrix over the entire cover.
Slip immediately after place the six centimeter dish containing the cover slips in the 15 centimeter dish. Transfer the dish to the incubator at 37 degrees Celsius with 5%carbon dioxide for 15 to 20 minutes. After the matrix has solidified, gently add five milliliters of complete medium to each six centimeter dish and push down any floating cover slips with the pipette tip then incubated for 24 hours at 37 degrees Celsius with 5%carbogen to allow the neuroblast to migrate out of the cell aggregates.
In this figure, the isolated rat RMS cells are shown to be immuno positive for the migratory neuroblast markers DCX and beta three tubulin. And this figure shows that the migratory neuroblast markers DCX and PSA NCA are expressed in the cells migrating out of the mouse. RMS Explan for mouse Neuroblast Nu nuclear affection, dissociated mouse RMS Neuroblast were nucle affected with GFP re aggregated embedded in a three-dimensional matrix and allowed to migrate for six hours.
The neuroblast migrating out of a re aggregated cell cluster show high transfection efficiency. Here, rat neuroblast were nucle affected with two different plasmids encoding GFP re aggregated embedded in matrix and left to migrate for 24 hours. The cells were then fixed and immunostain for GFP and beta three tubulin to measure the migration distance.
The aggregated cell cluster is divided into six equal sectors and the distance between the edge of the cluster and the furthest migrated cell is measured for each sector. This figure shows the quantification of relative distance migrated by the GFP positive NUCLE affected cells and control the GFP negative non nucle affected cells to monitor neuroblast migration. After S-H-R-N-A Nucle affection rat neuroblast were nucle affected with a control S-H-R-N-A vector or the same vector containing an HRNA target fasten and actin bundling protein cells were re aggregated over 48 hours embedded in matrix and left to migrate for 24 hours.
Aggregates were then fixed and immuno stain for GFP and beta three tubulin. Effective fasten depletion can be detected 50 hours after S-H-R-N-A nuclei affection by Western Blood Analysis and Acton is shown here as a loading control. Here is the quantitative analysis of the relative migration distance showing that fastened depletion significantly impairs neuroblast migration.
Following this procedure, other methods like postnatal electroporation can be performed to validate in vivo the results obtained with the NU Nuclear Affection migration assay.
