Plasmid-Mediated Generation of Recombinant Rotavirus in Cultured Cells

Zusammenfassung

Source: Philip, A. A., et al. Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins. J. Vis. Exp. (2020)

This video demonstrates the recovery of recombinant rotavirus by transfecting plasmids encoding viral genome segments into hamster kidney cells, followed by amplification in monkey epithelial cells, enabling studies of rotavirus biology, replication, and vaccine development.

Protokoll

1. Generation of recombinant virus
NOTE: Human and animal rotavirus research, including the generation and characterization of recombinant rotavirus strains, must be handled under Biosafety Level 2 (BSL-2) conditions and will require prior approval by the Institutional Biosafety Committee (IBC). Appropriate BSL-2 laboratory conditions are described in Biosafety in Microbiological and Biomedical Laboratories (BMBL) produced by the Centers for Disease Control and Prevention (CDC).

1. Day 1: seeding of BHK-T7 cells into 12-well plates
   1. Rinse a freshly confluent monolayer of BHK (baby hamster kidney)-T7 cells contained in a T75 flask 2x with PBS (phosphate-buffered saline). Disrupt the cell monolayer with trypsin-EDTA (ethylene diamine tetraacetic acid) solution and resuspend the cells in 5 mL of GMEM (Glasgow minimum essential medium) complete medium.
   2. Use an automated cell counter and trypan-blue solution to determine the concentration of viable BHK-T7 cells in the medium. Seed cells into 12-well cell culture plates, with each well containing 2 x 10⁵ cells in a total volume of 1 mL of GMEM (G418-free) complete medium. Incubate in a 37 °C, 5% CO₂ incubator. The cells should reach 80−90% confluency by day 2.
2. Day 2: transfection of BHK-T7 cells with plasmid mixtures
   1. To prepare the plasmid mixture, combine the following in a 0.5 mL microcentrifuge tube using plasmid stocks adjusted to 1 mg/mL: 0.8 µL each of SA11 pT7 plasmids VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP3, and NSP4, 2.4 µL each of SA11 pT7 plasmids NSP2 and NSP5, and 0.8 µL of pCMV/NP868R. Gently mix plasmids by tapping the tube and collect contents by pulse centrifugation. To prepare recombinant viruses expressing FPs, replace pT7/NSP3SA11 with the appropriate pT7/NSP3-2A-3xFL-FP plasmid.
      NOTE: Plasmid mixtures should be stored on ice until used. A separate tube should be prepared for each transfection.
   2. To prepare the plasmid/reduced serum medium/transfection reagent mixture: Add 110 µL of prewarmed (37 °C) reduced serum medium (Table of Materials) to each plasmid mixture and mix by gently pipetting up and down. Afterwards, add 32 µL of transfection reagent (Table of Materials) to each plasmid/reduced serum medium mixture; this yields a concentration of 2.5 µL of transfection reagent per µg of plasmid in mixtures. Vortex mixtures gently and incubate at room temperature for 20 min.
   3. During the 20 min incubation period, rinse BHK-T7 cells in 12-well plates (prepared on day 1) once with 2 mL of GMEM incomplete media. Afterwards, add 1 mL of SMEM incomplete medium to each well and return plates to the incubator.
   4. After the 20 min incubation period, add the plasmid/reduced serum medium/transfection mixture drop-by-drop to each well of the 12-well plates using a 200 µL pipettor. Gently rock plates and return to a 37 °C, 5% CO₂ incubator.
3. Day 4: overseeding transfected BHK-T7 cells with MA104 cells
   1. Rinse a freshly confluent monolayer of MA104 cells contained in a T75 flask 2x with PBS. Disrupt the monolayer using trypsin-EDTA solution and resuspend cells in 5 mL of DMEM complete medium.
   2. Using an automated cell counter and trypan-blue solution, determine the concentration of viable MA104 cells in the medium. Adjust the concentration to 8 x 10⁵ MA104 cells/mL in DMEM incomplete medium and add 0.25 mL of suspended cells (2 x 10⁵ cells) dropwise to the wells containing transfected BHK-T7 cells.
   3. Adjust the concentration of trypsin (porcine pancreatic type IX) in the medium to ~0.5 µg/mL by adding 0.8 µL of 1 mg/mL trypsin stock to each well.
      NOTE: Trypsin stocks should be prepared in PBS, aliquoted, and stored at -20 °C.
   4. Use the remaining MA104 cells to prepare 6-well plates that will be needed on day 7 to amplify recombinant viruses. To seed 6-well plates, dilute resuspended MA104 cells to a concentration of 1.5 x 10⁵ cells/mL in DMEM complete medium and place 2 mL in each well. Place plates in a 37 °C, 5% CO₂ incubator.
4. Day 7: recovery and amplification of recombinant virus from transfected cells
   1. Subject BHK-T7/MA104 cells in 12-well plates to three cycles of freeze-thaw under sterile conditions, moving plates between -20 °C freezer and a room temperature surface. After transferring lysates to 1.5 mL tubes, centrifuge tubes for 10 min at 500 x g (4 °C) to pellet large cellular debris. Collect supernatant and store at 4 °C (short term) or -20 °C (long term).
   2. Wash MA104 monolayers in 6-well plates (prepared on day 4) 2x with PBS. Place 2 mL of DMEM incomplete medium in each well that contains 0.5 µg/mL trypsin. Add 300 µL of supernatant recovered from BHK-T7/MA104 cell lysates into wells and place plates in a 37 °C, 5% CO₂ incubator.
   3. Incubate plates for 7 days or until complete cytopathic effects (CPE) are observed. Lyse MA104 cells in 6-well plates by three cycles of freeze-thaw, then transfer lysates to 1.5 mL microcentrifuge tubes. Pellet large cellular debris by centrifugation for 10 min at 500 x g (4 °C). Transfer the clarified cell lysates into 1.5 mL microcentrifuge tubes and store at -20 °C.

Materialien

Liste der in diesem Artikel verwendeten Materialien

Name	Unternehmen	Katalognummer	Kommentare
Baby Hamster Kidney - T7 RdRP (BHK-T7) Cells
Disposable Transfer Pipettes, Ultrafine Extended Tips	MTC Bio	P4113-11
Dulbecco's Modified Eagle Medium (DMEM)	Lonza	12-604F
Eagle's Minimal Essential Medium, 2x (2xEMEM)	Quality Biological	115-073-101
Ethanol, Absolute (200 proof)	Fisher Bioreagents	BP2818-500
Ethidium Bromide Solution (10 mg/ml)	Invitrogen	15585-011
Fetal Bovine Serum (FBS)	Corning	35-010-CV
Fetal Bovine Serum (FBS), Heat Inactivated	Corning	35-011-CV
Flag M2 Antibody, Mouse Monoclonal	Sigma-Aldrich	F1804
GenEluate HP Plasmid Midiprep Kit	Sigma	NA0200-1KT
Glasgow Minimal Essential Medium (GMEM)	Gibco	11710-035
Minimal Essential Medium -Eagle Joklik's Forumation (SMEM)	Lonza	04-719Q
Monkey Kidney (MA104) Cells	ATCC	ATCC CRL-2378.1
NanoDrop One Spectrophotometer	ThermoScientific
Opti-MEM Reduced Serum Medium	Gibco	31985-070
Phosphate Buffered Saline (PBS), 10x	Fisher Bioreagents	BP399-20
Porcine Trypsin, Type IX-S	Sigma-Aldrich	T0303
PureYield Plasmid Miniprep System	Promega	A1223
Qiagen Plasmid Maxi Kit	Qiagen	12162
Qiagen Plasmid Midi Kit	Qiagen	12143
QIAprep Spin Miniprep Kit	Qiagen	27104
SA11 pT7 Transcription Vectors	Addgene	89162-89172
SA11 pT7/NSP3 Transcription Vectors Expressing Fluorescent Proteins
Trypan blue	Corning	25-900-CI
Trypsin (0.05%)-EDTA (0.1%) Cell Dissociation Solution	Quality Biological	118-087-721