The overall goal of this procedure is to virally, manipulate oligodendrocyte precursor cells or OPCs to over express proteins of interest. For the in vitro study of CNS Myelination Nation, this is accomplished by first cloning a lentiviral vector. The next steps are to produce the lentivirus and determine the optimal viral concentration for the experiment.
Following this dissected and cultured OPCs are transduced with the lentivirus. The final step is to seed the transduced OPCs onto cultured neurons dissected from the DRG. Ultimately, the myelinating co cultures are assessed for myelin protein expression by western blot analysis and visualized for the formation of myelinated axonal segments by immuno staining.
The main advantage of this technique over existing methods, such as pharmacological tools, is that the lentiviral approach allows us to selectively overexpress either wild type or mutant proteins in our desired cell type, such as in this case, oligodendrocytes In preparation clone the required 2K seven lent vectors as described in the text protocol in the morning of the day of transfection, prepare 32 million HEK 2 93 T cells in a T 1 75 flask containing 25 milliliters of media. Critically, these cells must be stuck down before transfection, so plate them sooner if needed. In a 50 milliliter Falcon tube, prepare a master mix for the T 1 75 flask transfection as detailed in table one, add DNA to warm DMEM vortex, then add sterile PEI last to avoid premature precipitation.
Mix well by inverting the tube three or four times, then incubate the mix for about 15 minutes on the bench. Change the media on the cells and then add the transfection mixture. DROPWISE gently mix the media over the cells and then incubate the cells overnight.
The following day. GFP expression can be checked by fluorescence microscopy. 48 hours after the transfection, collect the first batch of viral supernatant and add 25 milliliters of fresh media to the cells.
Centrifuge the viral supernatant at 1, 140 Gs for 10 minutes at four degrees Celsius. Transfer the viral supernatant without the pellet of cell debris to a new tube and store it at four degrees Celsius. The next day at 72 hours post transfection, collect the last batch of viral supernatant centrifuge to remove the cell debris and pool with the first batch of cleared viral supernatant.
Then concentrate the virus by ultra centrifuging the viral supernatant at 170, 000 Gs for 90 minutes. At four degrees Celsius. Unload the ultra centrifuge and pour away the supernatant, leaving a palate of precipitated PEI and the virus, which is not visible at the bottom of the 30 milliliter tube.
Repeat this process. Centrifuging all the viral supernat in batches in the same 30 milliliter tubes to concentrate the virus to resuspend the virus in each ultracentrifuge tube. Add 500 microliters of Sato media and cover the tube with film.
Then vortex the tube for 30 seconds and mechanically dislodge the pellet with a pipette tip. Repeat this vortex dislodged cycle about six times to ensure the pellet is fully suspended into the Sato media. Now pool all the resuspended virus in Sato in a fresh micro tube.
Briefly spin down the pooled supernatant at maximum speed for one minute. To pellet the PEI then collect the supernatant containing the virus and filter by passing the solution through a 0.45 micron filter using a syringe to wrap up. Aliquot the purified virus in Sedia into 2050 and 100 microliter volumes and store them all at negative 80 degrees Celsius.
For this protocol, have primary oligodendrocyte precursor cells. OPCs cultured in 10 centimeter plates with 10 milliliters of sedia containing the following growth factors, C-N-T-F-P-D-G-F-N NT three, and for the cells must have been cultured for 24 hours under 8%Carbon dioxide. Begin with aspirating the media and remove it completely.
Then replace it with 10 milliliters of the same media with the growth factors. Infect your OPCs with lentivirus, expressing the protein of interest by adding the required volume of virus dropwise into the media. Also in a separate OPC plate, mix the control lentivirus that expresses only GFP into the media culture, the cells for another 48 hours.
Then collect the OPCs from the plates for seeding onto the cultured GRG neurons. First rinse each plate with eight milliliters of EBSS twice. Make up one milliliter of trypsin EDTA 0.05%with nine milliliters of warm EBSS.
Add five milliliters of the diluted trypsin to each plate. Incubate at 37 degrees Celsius for up to two minutes. Neutralize the trypsin by adding five milliliters of 30%FBS in EBSS.
Then using a 10 milliliter pipette. Wash the OPCs from the plate surface. Turn the plate by a quarter turn each time to collect all the cells.
Transfer the cell suspension to a 15 milliliter tube. Centrifuge the cells at room temperature for 15 minutes at 180 Gs.Now aspirate the supernatant and resus. Suspend the cell pellet in a milliliter of warm saddo media without growth factors and count the cells.
Next, prepare the DRG neurons that are cultured on the 22 millimeter cover slips by completely aspirating the media from their plate. Then gently seed 200, 000 OPCs dropwise to each cover slip. The seeding volume must be less than 200 microliters per cover slip.
Let the cells settle without moving the plate After 10 minutes, top each well with a milliliter of warm s omed. Now replay the remaining sister OPCs. With s omed containing growth factors, they can be used later to verify expression of the protein of interest.
A flag tagged extracellular signal related kinase. One construct was used for lentivirus production restriction Enzymes were used to verify the packaging construct and the accessory construct to determine the optimum virus dilution to use on the OPCs. The IR one lentivirus was titrated in HEK 2 93 T cells at a range of viral dilution to HEK 2 93 T cells.
Expression levels of the gene of interest increased with time. Next, purified OPCs were infected and cultured for at least two days to ensure transgene expression. FLAG and GFP levels were confirmed in Sister OPCs.
The OPCs were then seated onto DRG neurons. The OPCs then differentiated into oligodendrocytes, and as assessed by westerns, they began to express myelin proteins. Some mature oligodendrocytes myelinated, which was visualized by MBP staining axons were stained by neurofilament or beta three tubulin to ensure that the axon densities were taken into account.
When analyzing myelination levels After seeding the OPCs, it's important to culture. The sister APCs. This way, the protein of interest or the tag on the protein of interest, that expression can be verified in the sister APCs if it can't be verified in the co-culture.
