1. Cloning of 2K7 Lentivector
- Before cloning the gene of interest into the 2K7 lentiviral vector, subclone the gene into the pENTR vector (3637 bp, Kanamycin resistant) using standard molecular techniques. Use the EcoRI and SacII restriction sites for the subcloning.
- Amplification of the 2K7 Lentivector
- Transform 2K7 lentivector DNA by gently mixing 100 ng of plasmid DNA with competent cells, and incubate on ice for 30 min.
- Heat shock DNA/ competent cells mix at 42 °C for 90 sec and plate them on LB-Agar plates containing both ampicillin (100 µg/ml) and chloramphenicol (15 µg/ml) at 37 °C for 16-18 hr.
NOTE: Chloramphenicol is used to prevent recombination between the long terminal repeats.
- The next day, grow selected bacterial clones in Luria-Bertani (LB) media containing both ampicillin (100 µg/ml) and chloramphenicol (15 µg/ml) at 37 °C for 16-18 hr. Extract the DNA using a commercial plasmid DNA Maxiprep kit as per the manufacturer's instructions.
- Sub-cloning from pENTR vector to 2K7 Lentivector (Figure 1).
- Add the following to a 1.5 ml tube and mix gently: L4-R1 pENTR-pDNOR-CMV (promoter) (60 bng)1-2.5 µl L1-L2 pENTR4IRES2GFP (with gene of interest) (80 bng)1-2.5 µl K7 lentivector (80 ng/µl)1 µl TE-buffer, pH 82-5 µl.Total 8 µl.
- Remove the clonase enzyme mix from -80 °C and thaw on ice for 2 min. Ensure that this enzyme is a fresh aliquot from -80 °C as the enzyme has substantially reduced cloning efficiency with repeated freeze-thaw cycles
- Add 2 µl of enzyme per reaction and mix well via vortex, and incubate the clonase reaction mixture at 23-25 °C for 6-24 hr.
- Add 1 µl of proteinase K solution (supplied with enzyme kit) to the clonase reaction mixture and incubate for 10 min at 37 °C.
- Transform the clonase reaction mixture into competent cells and grow the select colonies in LB media containing ampicillin (100 µg/ml) at 37 °C for 16-18 hr.
- Extract and purify DNA using a commercial plasmid DNA Miniprep kit as per the manufacturer's instructions.
- Confirm the DNA via digestion using restriction enzymes Spe I and Sac II and an appropriate buffer as per the manufacturer's instruction to remove the fragment containing the promoter and gene of interest.
NOTE: This step is critical to check if the subcloned DNA has the correct insert gene of interest with the right vector backbone. The size of the vector itself without the inserted fragment is 6.7 kb. The size of the released inserted fragment includes both the promoter and the insert gene of interest. Calculate the expected fragment sizes from the digest for the specific gene via a DNA sequence editing program, e.g., APE-A Plasmid Editor.
- Check the sizes of both the DNA vector backbone and the released fragment by running a 1% agarose gel in 1x TAE buffer (see Table 1) at 100 V.
- Amplify the confirmed DNA by growing bacteria in 500 ml of LB media containing ampicillin (100 µg/ml) at 37 °C for 16-18 hr.
- Extract and purify DNA using a commercial plasmid DNA Maxiprep kit as per the manufacturer's instructions.
NOTE: Maxiprep typically generates a sufficient amount of DNA required for viral production.
- Repeat steps 1.3.9-1.3.10 to amplify the following DNAs for lentiviral preparations: Envelope vector (PMD2.G, 6.1 kb), Package vector (pBR8.91, 12.5 kb), and an empty lentiviral vector as a control (GFP-CMV-2K7, 8.7 kb).
2. 2K7 Virus Production
NOTE: Day 1:
- On the day of transfection, plate 32 million HEK293 T cells in a T175 flask containing 25 ml Human Embryonic Kidney (HEK) 293 T cell media (see Table 1). Alternatively, plate 16 million cells the day before the transfection if time runs tight on the day of transfection.
NOTE: Transfection can be equally successful by plating cells on the day of transfection or prior to the day. Using either of the two alternatives, the critical point here is to make sure cells are stuck down on the culture dish surface prior to transfection.
NOTE: Day 2:
- Transfection
- Prior to transfection, dilute DNA to 1 µg/µl in Tris-ethylenediaminetetraacetic acid (TE) Buffer that contains 10 mM Tris pH 8, 1 mM EDTA pH 8 in deionized water.
- In a 50 ml tube, prepare a master mix (Table 2) for transfection in the T175 flask. Add DNA to pre-warmed Dulbecco's Modified Eagle Medium (DMEM) and mix well by vortex, then add sterile polyethyleneimine (PEI) (see Table 1 to avoid premature precipitation.
- Mix well by inverting the tube 3-4x, and incubate for 15 min at room temperature (RT).
- Perform a full media change on HEK293T cells. Aspirate off the culture media from cells completely and feed with pre-warmed HEK293T cell culture media (25 ml per T175 flask).
- Add the transfection mixture (DNA/PEI mixture) dropwise to the monolayer cells. Move gently to mix well and incubate transfected cells at 37 °C, 5% CO2, overnight.
NOTE: Day 3:
- To ensure the transfection is successful, check green fluorescence protein (GFP) expression 24 hours post-transfection via fluorescent microscopy.
NOTE: Over 50% of cells expressing GFP typically indicate a good transfection.
NOTE: Day 4:
- At 48 hr post-transfection, collect the viral supernatant and replace it with fresh HEK293 T media (25 ml per T175 flask).
- Centrifuge the viral supernatant at 1,140 x g for 10 min at 4 °C to clear up cell debris from the supernatant. Transfer the cleared supernatant to a 50 ml tube and store it at 4 °C.
NOTE: Day 5:
- At 72 hours post-transfection, collect the second batch of viral supernatant. Repeat step 2.3.1 and pool 48 and 72 hr cleared supernatants.
- To concentrate the virus, centrifuge viral supernatant at 170,000 x g for 90 min at 4 °C using 30 ml ultracentrifuge tubes.
- Discard the supernatant and repeat step 2.6 until all the cleared supernatant has been centrifuged leaving an (invisible) pellet of virus plus precipitated PEI in the base of the tube.
- To resuspend the virus, add 500 µl SATO media (see Table 1) to the ultracentrifuge tubes. Vortex for 30 sec and scrape the base of the tube with a pipette tip to mechanically loosen the virus. Repeat this step 6x in order to lose the viral pellet.
- Pool the resuspended virus into microcentrifuge tubes and spin very briefly to remove insoluble PEI. Filter the supernatant through a 0.45 µm filter to remove proteins.
- Aliquot the virus into 20 µl, 50 µl, and 100 µl aliquots and store at -80 °C.
3. Transducing OPCs
- Culture primary OPCs in SATO media (10ml/10cm plate) with Ciliary neurotrophic factor (CNTF, 10 ng/ml), Platelet-derived growth factor (PDGF, 10 ng/ml), Neurotrophin 3 (NT3, 1 ng/ml), and forskolin (4.2 µg/ml) at 37 °C, 8% CO2 for 24 hr after dissection.
- Completely aspirate off the OPC culture media, and feed cells with freshly made SATO media (10 ml) with growth factors (see above in step 3.1).
- Add virus to the OPCs to the optimal concentration, culture OPCs for a further 48 hr.
4. OPC Seeding for Myelinating Co-cultures (Figure 2)
- To remove OPCs off the surface, first rinse OPC plates with 8 ml Earle's balanced salt solution (EBSS) twice, then incubate cells with 5 ml of warm 0.05% Trypsin-EDTA diluted 1:10 with EBSS at 37 °C for 2 min.
- Neutralize the trypsin with 30% FBS in EBSS (5 ml) and remove cells from the plate by pipetting. Transfer cell suspension to a 15 ml tube, and centrifuge at 180 x g for 15 min at RT.
- Aspirate off the supernatant and resuspend the cell pellet in 1 ml pre-warmed SATO media followed by cell count.
- Prior to the OPC seeding, completely aspirate media from the DRG culture plate. Gently seed 200,000 OPCs dropwise onto the DRG neurons as previously described.
NOTE: The total OPC seeding volume must be less than 200 µl per 22 mm coverslip.
- Leave cells to settle without moving the plate for 10 min in the tissue culture hood, then gently top up with 1 ml pre-warmed SATO media per well.
- Replate the remaining sister OPCs with SATO media with growth factors (see step 6.1). Use these sister OPCs to verify the expression of the protein of interest.
- After 24 hr, replace the SATO media with the co-culture media (2 ml/well) containing SATO media (no factors) and neurobasal (v/v) with 1% B2Maintain the co-cultures for 14 days with media change every 2-3 days.
- Assess the co-cultures for myelin protein expression by western blot analysis and visualize the formation of myelinated axonal segments by double-immunostaining with antibodies against myelin basic protein markers and neuronal markers.
