Production of rabies viral vectors

Production protocol for native coat and envA-pseudotyped CVS-N2c/SADB19 RVdG particles

Reagents:

   1. DMEM+:          430ml DMEM high glucose + Glutamax           

                               50ml FCS

                               5ml Pen/strep

                               5ml Na-pyrovate

                               5ml non-essential amino acids

       Filter sterilize; store at 4℃

   2. PEI 1mg/ml

   3. 20% Sucrose    40gr sucrose

                               11.7gr NaCl

                               4ml HEPES 1M, pH7.4

                               1ml EDTA 0.5M, pH7.9 

        Volume to 200ml; filter sterilize; store at 4℃

Cell lines:

1. HEK293BGT:  B19_oG-IRES-Puro + oT7pol-IRES-BSD
2. BHKeT:   envA-IRES-Neo + TVA-IRES-Puro
3. HEK293-TVA: TVA-IRES-Puro

Day 1:    

Resuspend HEK 293BGT cells from a fully confluent 10cm plate in 10 ml fresh DMEM+ and re-plate 1 ml from the suspension into a six well plate (1ml per well X number of different viruses to be rescued).   
 

Day 2: 

At this stage cells in each well should be 80-90% confluent. 

1. For each plate, prepare the following cocktail:

                Vial 1:    90 µl DMEM (without FBS)

                             10 µl PEI 1mg/ml

                              Vortex for 15 sec.

                Vial 2:   2 µg vector

                            0.5 µg pTIT-N

                            0.5 µg pTIT-L

                            0.5  µg pTIT-P

                            0.5  µg T7pol (optional, speeds up rescue process)

                            *DMEM – add to 100 µl

     2. Add the content of vial 2 to vial 1 and vortex briefly. Incubate for 15-20 min at RT.

     3. Add the entire transfection reaction to a single well. 

     4. After 5-8 hours replace the medium to fresh 2 ml DMEM+.

Day 3:

Resuspend the cells in the transfected well and plate them all in a 100 mm culture plate. Bring the final DMEM+ volume to 10 ml. 

Day 5:

Replace the medium in the plate with 10 ml fresh DMEM+ and inspect the plates daily for fluorescence. It should already be visible 4-5 days after transfection. Replace the medium daily until >90% of the cells are fluorescent, usually 1-3 days after fluorescence was first detected. At that point, collect all the medium from the plate and filter sterilize it and store at +4℃ for up to two weeks, or at -80℃ for longer.

To continue with the protocol, split BHKeT cells from a confluent 10cm plate in a ratio of 1:10 into two new 10 cm plates.

Day 6:  

Use the collected native-coat supernatant to transduce BHKeT cells by adding to each plate 100-500 µl of the native-coat supernatant.

Day 7:

Fluorescence should be visible at this point, even if only sparsely. 

Wash the medium from the transduced BHKeT cells 1-2 times to get rid of remaining NC vectors, resuspend the cells from each of the two transduction plates using trypsin and replate them into two new 150 mm plates.

Day 10:

At this point, the plates should be fully confluent and >80% of cells should be labeled. Collect the entire medium from each plate and replace with fresh medium (ideally 15 ml per plate). Pool and filter the collected supernatant through a 0.45 or 0.22 µm filter and store at 4℃. Repeat this process daily for three or more days.

Plate HEK293-TVA cells in low confluence (~50,000 cells per well in a 6well plate). Allocate 3 wells of HEK293-TVA cells and 1 naïve HEK293-T cells for each vector produced.

Day 12:

1. Pool all of the collected virus-containing supernatant and distribute into ultracentrifuge tubes. To the bottom of each centrifuge tube, gently add the 20% sucrose solution to a final volume of 10% of total medium volume.
2. Centrifuge at 70,000rcf for 90 minutes at 10℃. Discard of the supernatant and resuspend the viral pellet (usually invisible) with 500 µl filtered PBS and transfer between all centrifuge tubes until all the virus has been resuspended. Transfer to a 1.5 ml Eppendorf and store at 4℃ between cycles if multiple centrifugation cycles are needed.
3. Repeat steps 1+2 and resuspend after each cycle with the virus collected in the previous cycles until all the virus-containing medium has been exhausted.
4. Aliquot the resuspended virus into 200 µl PCR tubes – 5 µl virus per tube and store at -80℃ until use.

Titration:

1. Mix 1 µl of the concentrated, psuedotyped vector and mix it in 1 ml DMEM in a 1.5 ml Eppendorf and vortex well.
2. Use 100, 10 and 1 µl of the diluted virus to transduce the 3 HEK293-TVA wells and the remaining 900 µl to transduce the naïve HEK293-T cells, to monitor for contamination with native coat particles. Calculate the titer according the formula published in Wickersham et al., 2010.

Full article (for citation)

Anton Sumser, Maximilian Joesch, Peter Jonas and Yoav Ben-Simon (2022) Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling eLife 11:e79848.

https://doi.org/10.7554/eLife.79848