Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 119

(i) Pipette 0.5 mL of the 10 7 cells/mL suspension in the 0.4-cm
cuvette. Then add to the cuvette:

-10 L of the recombinant sindbis vector RNA produced

before.
-10

μ

L helper RNA produced before mix cells and RNAs
by tapping the cuvette gently.

μ

(j) Electroporate cuvette: Voltage setting on Biorad Gene Pulser
XCell should be
Exponential protocol:

1. V: 1,200 V.
2. C: 25 F.

3. R: infi nite.
4. C: 0.
Pulse once.
After each electroporation the time constant displayed should
be ~0.6-1.0 ms. These settings will need to be optimized if
using a different size of cuvette or a different electroporator.
After electroporation, the end of the procedure needs to be
performed in a biosafety level 2 tissue culture room.
(k) Place cuvette back on ice to minimize damage to electropor-
ated cells. Add 500

μ

MEM + FBS to resuspend cells in the
cuvette (total volume should be ~1 mL including cell suspen-
sion). Transfer this 1 mL suspension to the 150-mm 2 fl ask
containing 11 mL

μ L α

MEM + FBS prepared in step (h). The
total volume of virus suspension should be 12 mL.
(l) Incubate cells for ~36 h at 37 °C.
(m) Check the effi ciency of transfection after ~18 h by calculating
the percentage of GFP-expressing cells (if the insert was
cloned in the pSINRep(nsP2S 726 )-IRES-GFP). A minimum
of 60 % transfection effi ciency is necessary to produce a good
titer of virus compatible with in vivo infection experiments.
One hundred percent transfection effi ciency can be observed
within 36 h if the protocol is optimized. At this point the
cultures produce a very high number of infectious viral parti-
cles; therefore, the experimenter must be careful handling
them.

α

We are now working with infectious virus particles. It is crucial to
wear protective clothing and perform all collection and concentration
procedures in a level 2 biosafety area. All liquid and solid materials
that came into contact with the viral solution must be inactivated
with hypochlorite before disposal.

Collecting Virus
and Concentration
for In Vivo Injection

(a) Cool down the Beckman ultra-centrifuge and SW40Ti swing-
buckets rotor to 4 °C.