Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 99

and resuspend cell pellet in a small volume (<5 mL) of PBS. Add
PBS to 25 mL and recentrifugate for 5 min at 800× g . Resuspend
cells well in approximately 2.5 mL PBS per T175 fl ask, equivalent
to 1-2 × 10 7 cells per mL. Use cells immediately for electroporation
(shorter storage (<1 h) on ice is acceptable). Transfer 0.4 mL
BHK-21 cell suspension to 0.2 cm cuvettes or 0.8 mL to 0.4 cm
cuvettes. Add in vitro transcribed recombinant RNA (20-45 μ L)

and helper RNA (20 L) to the cell suspension. Apply two con-

secutive pulses. The following settings should be applied for the
BioRad Gene Pulser:

μ

0.2-cm Cuvette 0.4-cm Cuvette

Capacitance extender 960 μ F 960 μ F

Voltage 1,500 V 850 V

Capacitor 25 μ F 25 μ F

Resistance (pulse controller) ∝ Ω Disconnected

Expected time constant (tc) 0.8 s 0.4 s

The BioRad Gene Pulser II requires the following modifi cations:

The pulse controller should be set to “high range” and “ ∝ ”

●

The capacitance rotary switch should be set to “high
capacitance”

●

The following settings should be applied: 360 V and 75 μ F

●

The obtained resistance for 0.2 cm cuvettes is 10 Ω and the

●

time constant 0.7-0.8 s

Dilute the cells immediately 25-fold in cell culture medium
and transfer cells to T fl asks or plates. Incubate cells at 37 °C
overnight in an incubator with 5 % CO 2 .

3.5 Lipid-Mediated
Transfection of RNA

Alternatively to electroporation transfection reagents can be
applied for virus production. DMRIE-C (Invitrogen) and other
transfection reagents have been used for BHK-21, COS7, and
CHO-K1 cells. Plate 1.5-3 × 10 5 BHK-21 cells in 35-mm Petri
dishes or on 6-well plates and culture the cells to approximately
80 % confl uency. Wash cells with Opti-MEM I reduced-serum
medium (Gibco BRL). Prepare the following cationic lipid-RNA
complexes: add 0, 3, 6, 9, 12, and 15

L of DMRIE-C to the six
1.5-mL microcentrifuge tubes with 1 mL Opti-MEM I reduced-
serum medium at room temperature and vortex briefl y. Mix 10

μ

μ L

(~5 g)

helper RNA and add the mixture to each tube and vortex briefl y.
Add the lipid-RNA complexes immediately to the washed cells and
incubate at 37 °C for 4 h. Replace the transfection medium with
prewarmed (37 °C) complete BHK medium and incubate the
BHK cells at 37 °C overnight in an incubator with 5 % CO 2 .

μ g) in vitro transcribed recombinant RNA and 5 μ L (~2.5 μ