Jun 04, 2026

RSV Reverse Genetics

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjnnxbgk5/v1
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjnnxbgk5/v1
Protocol Citation: Carolina Lopez 2026. RSV Reverse Genetics. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnnxbgk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 29, 2024
Last Modified: June 04, 2026
Protocol  Integer ID: 100887
Keywords: recombinant rsv from plasmid, rsv reverse genetics this protocol, producing recombinant rsv, rsv reverse genetics, plasmid
Abstract
This protocol describes the procedure for producing recombinant RSV from plasmids
Materials
  • BSR-T7/5 cells (It is better to use the cells within Passage 4)
(BSR-T7/5 cells were kindly provided by Dr. Conzelmann. Reference: https://doi.org/10.1128/jvi.73.1.251-259.1999)
  • Opti-MEM (Gibco/Life Technologies catalog #11058-21)
  • Cell culture Growth Media: Tissue Culture medium (see recipe below) + 500 μg/mL of Geneticin (Thermo Fisher, 10131035)
Equivalent Plasmid with RSV antigenome (NR-36460, BEI Resources)
  • Helper Plasmids – (all codon optimized):
pA2-Lopt, L protein (NR-36461, BEI Resources)
pA2-Nopt, N protein (NR-36462, BEI Resources)
pA2-Popt, P protein (NR-36463, BEI Resources)
pA2-M2-1opt, M2-1 protein (NR-36464, BEI Resources)
  • Lipofectamine 2000 transfection reagent (Gibco/Life Technologies catalog #11668-019)
  • Phosphate buffered saline 7.2 (Gibco/Life Technologies catalog #20012027)
  • 6-well/ 25 cm2 tissue culture plates/flasks
  • Shaker/rocker plate
  • Tissue culture humidified incubator with 5% CO2, 37ºC
  • Assorted sterile pipettes and tips



TISSUE CULTURE MEDIUM (TCM): Filter through 0.2 µm filter
ComponentAmountConc. Supp.Product information
DMEM500 mLGibco Cat. # 11965092 (#11965118-cs)
Gentamicin500 µL 50µg/mLGibco Cat #15750060 (#15750078-pk) – 50 mg/mL
Sodium Pyruvate 5.0 mL1mMCorning Cat #25-000-Cl – 100mM
L-Glutamine 5.5 mL 2 mMSigma Aldrich Cat #G7513 – 200mM
FBS50 mL10%

INFECTION MEDIUM – RSV: Filter through 0.2 µm filter
ComponentAmountConc. Supp.Product information
DMEM500 mLGibco Cat. # 11965092 (#11965118-cs)
Gentamicin500 µL 50µg/mLGibco Cat #15750060 (#15750078-pk) – 50 mg/mL
Sodium Pyruvate 5.0 mL1mMCorning Cat #25-000-Cl – 100mM
L-Glutamine 5.5 mL 2 mMSigma Aldrich Cat #G7513 – 200mM
FBS10 mL2%

Before start
Updated 06/2026 by SF
RSV Reverse Genetics
2h 37m
This protocol is adapted from Stobart, et al. 2017 and BEI Resources (see References tab)

Note: This protocol assumes the user is familiar with cell culture techniques and transfection procedures.

It’s always a good idea to use pSynkRSV(mKate2) plasmid as a positive control if rescuing adapted strains (Confirm these plasmids with someone who knows them, or verify by whole plasmid sequencing before you use them).

Initial cell culture:
  1. Routine sub-passage BSR T7/5 cells in tissue culture medium +Geneticin (500 μg/mL) as a growth medium. Add the Geneticin (500 μg/mL) to the growth medium every passage.

Prepare 6 well plates for transfection from T75 flask one night before transfection.
  1. Aspirate the growth medium from the flask.
  2. Add 2 mL of trypsin-EDTA and rock flasks to distribute the trypsin-EDTA and incubate at 37 °C 5% CO2 for about 00:02:00 .
  3. When cells start to dislodge from the flask, add 8 mL of tissue culture medium to the flask and use a pipet to suspend the cells in this growth medium.
  4. Count the cells and put 4.5x105 cells in one well of the 6-well plate. Incubate the plates at 37 °C 5% CO2 in the tissue culture incubator.
2m
The next day, check the cells for a confluent monolayer. Then prepare the reagents for the transfection procedure.

Transfection will be with Lipofectamine 2000 as the transfection reagent. Additionally, it is important to include control transfections (a negative control: Lipofectamine only - no DNA and a positive control: wild type virus for mutants or another positive transfection control)
1. Use a 3:1 ratio of Lipofectamine (μL) to plasmid/helper plasmid (μg).
2. Dilute each component with Opti-MEM to make 100 µL of each. After dilution, allow each dilution to sit at room temperature for 00:05:00 . Use the following amounts of each component per transfection:
a) RSV antigenome (NR-36460) 0.8 μg (8 μL of 0.1 μg/μL ) + 92 μL Opti-MEM
b) pA2-Lopt, L protein (NR-36461) 0.2 μg (2 μL of 0.1 μg/μL) + 98 μL Opti-MEM
c) pA2-Nopt, N protein (NR-36462) 0.4 μg (4 μL of 0.1 μg/μL) + 96 μL Opti-MEM
d) pA2-Popt, P protein (NR-36463) 0.4 μg (4 μL of 0.1 μg/μL) + 96 μL Opti-MEM
e) pA2-M2-1opt, M2-1 protein (NR-36464) 0.4 μg (4 μL of 0.1 μg/μL) + 96 μL Opti-MEM
f) Lipofectamine 2000 6.6 μL + 93.4 μL Opti-MEM
Note
For multiple transfections increase the above quantities proportionally.
3. After allowing the diluted components to sit at room temperature for 00:05:00 , combine all six components in one vial, mix gently and incubate the transfection mixture at room temperature for 00:20:00 .
4. Transfection mixtures should be 600 µL total (Opti-MEM, Lipofectamine, and DNA). Aspirate the media from the BSR T7/5 cell culture plate, wash cells twice with 1 mL Opti-MEM for each wash, and aspirate the final wash.
5. Add 600 µL transfection mixture to each well and incubate the plate for 02:00:00 at room temperature on a shaker/rocker plate set at low speed.
6. After 2 hours, add an additional 600 µL Opti-MEM per well and place the plate in a 37 °C 5% CO2 tissue culture incubator overnight (8-12 hours).
2h 30m
After overnight incubation
  1. Aspirate and discard the transfection mixture from the wells.
  2. Wash each well once with 1 mL sterile PBS.
  3. Aspirate the PBS and replace with 2 mL of infection medium - RSV per well (Note: no Geneticin is added in the medium post-transfection).
  4. Continue incubation at 37 °C 5% CO2 in the tissue culture incubator overnight.
Monitor flasks for cytopathic effect (CPE) and sub-pass transfected cells into T25/T75 flasks when confluent (approximately every 48 hours). CPE shows first as mini-syncytia and then grows into rounded up clumps of cells. Cells should remain in infection medium - RSV throughout the rest of the rescue.
When CPE is evident throughout >50% of the flask, which typically is ~7 days post transfection (mutants may require more or less time), scrape the cells into the growth media and aliquot into cryovials following these steps:
  1. Scrape the T75 flasks and transfer contents to 50 mL tube
  2. Spin at 1180 rpm for 00:05:00
  3. Remove the majority of the media and place on ice, resuspend the cell pellet in about 2.5 mL of the media
  4. Freeze/thaw pellet 3 times, with vortexing between each round
  5. Spin at 1180 rpm for 00:05:00
  6. Combine the media from the freeze/thaw with the media placed on ice
  7. Aliquot into tubes, snap freeze, and place at -80 °C or colder.
-This is P0 of the stock-
10m
Protocol references
Stobart, C.C., Hotard, A.L., Meng, J., Moore, M.L. (2017). BAC-Based Recovery of Recombinant Respiratory Syncytial Virus (RSV). In: Perez, D. (eds) Reverse Genetics of RNA Viruses. Methods in Molecular Biology, vol 1602. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6964-7_8

The following reagents were obtained through BEI Resources, NIAID, NIH:
- Bacterial Artificial Chromosome Plasmid pSynkRSV-I19F Containing Antigenomic cDNA from Respiratory Syncytial Virus (RSV) A2-Line19F, NR-36460
- Respiratory Syncytial Virus (RSV) A2 Large Polymerase (L) Helper Plasmid, pA2-Lopt, NR-3646
- Respiratory Syncytial Virus (RSV) A2 Nucleoprotein (N) Helper Plasmid, pA2-Nopt, NR-36462
- Respiratory Syncytial Virus (RSV) A2 Phosphoprotein (P) Helper Plasmid, pA2-Popt, NR-36463
- Respiratory Syncytial Virus (RSV) A2 Matrix 2-1 (M2-1) Helper Plasmid, pA2-M2-1opt, NR-36464

Download pSynkRSV-l19 product sheet from BEI.pdfpSynkRSV-l19 product sheet from BEI.pdf