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Biomedicum Genomics Oy
support@bmgen.fi
Haartmaninkatu 8
FI-00290 Helsinki
Finland
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Use of Recombinant Virus Products
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BMGen generally uses two methods for infection of target cells: a classic
transduction and a centrifugation-based spin transduction.
Below you can find a recombinant virus protocol suitable for transduction
of BMGen made micro-, mini-, midi- and maxiscale lentiviral as well as
ecotropic and amphotropic virusparticles.
Transduction of cells with recombinant lenti- and retroviral
particles
The following protocol is suitable for BMGen made mini, midi and maxiscale
lentiviral as well as ecotropic and amphotropic virusparticles. We
generally use two methods for transduction (=infection): classic
transduction and centrifugation-based spin transduction. Classic
transduction procedure is gentler to the cells than spin transduction.
However, spin transduction maximizes the infection efficiency by low-speed
centrifugation. The following examples are for 6-well or 10 cm petri dish
plates. Scale up or down according to your needs.
Classic transduction
Day 1:
Seed cells to the plates. As a gauge, we seed 150.000 mouse embyonic
fibroblasts (MEFs) on a single well of a 6-well plate.
Day 2:
• 6-well plates: Drain the media from the wells and replenish with 1 ml
virusparticles + 2 ml media per well and 8 µg/ml polybrene (use stock 8
mg/ml 1:1000).
• 10 cm plate: Use 2-4 ml virusparticles and total up to 6 ml with the
media.
Incubate for approximately 7 h (after 10 h polybrene is cytotoxic for some
cells). Remove the virus and feed cells with 3 ml (6-well plate) or 10 ml
(10 cm dish) of media to allow cells to recover overnight.
Day 3:
In the morning, split cells and seed e.g. 1:3, 1:30 and 1:300 in growth
medium. EGFP expression usually peaks around 24-48h after transduction. If
you aim to perform antibiotic selection, add antibiotics around 24h
post-infection (in the evening of day 3). Include a dish of uninfected
cells as a control for antibiotic killing.
Change medium every 2 or 3 days until the selection is complete.
Note 1. For amphotropic infections, test the target cell culture
supernatants for virus production (they should score negative) by reverse
transcriptase (RT) assay. BMGen does not provide RT assay services at
present. Testing for exclusion of replication competent virus (RCV),
needed to confirm the biosafety of the cell lines, is available as a
service.
Note 2. You aim to use minimal antibiotic concentration that kills all
uninfected cells. If no data available for your cells, establish a kill
curve.
Spin transduction
Day 1:
Seed example MEFs at density of 150.000 cells/well on 6-well plate.
Day 2:
• Drain the media from the wells and replenish with 1 ml virusparticles
+ 2 ml media per well and 8 µg/ml polybrene (use stock 8 mg/ml 1:1000).
• Incubate the cells with virusparticles for 10 min at 37C in cell
incubator, centrifuge the 6-well plate at RT, 2500 RPM for 30 min,
incubate again for 10 min at 37C and replace the virusparticles with 2 ml
of complete medium.
Day 3:
18 h-24 h later, harvest cells for assays.
Reagents
To make 8 mg/ml polybrene -> dissolve 80 mg/10 ml DDW, filtrate through
0.22 µM acrodiscs -> store at +4C.
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