Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 100

3.6 Harvest of
Recombinant Viral
Particles

Maximum production of recombinant SFV particles occurs within
the fi rst 24 h. The titers obtained are generally in the range of
10 8 -10 9 (occasionally 10 10 ) infectious particles per mL. Slightly
higher titers can be achieved by extending the incubation time to
48 h. Harvest virus by carefully removing the medium from the
BHK-21 cells. Cell debris and possible contaminants are removed by
fi lter sterilization through a 0.22- m fi lter (Millipore) ( see Note 5 ).

It is highly advisable to aliquot virus stocks as repeated cycles of
freezing and thawing can reduce the titers signifi cantly. Virus stocks
can be stored at −20 °C (for weeks) and at −80 °C (for years).

μ

Virus stock production from the conventional SFV helper vectors
renders directly infectious particles. However, packaging with the
second-generation pSFV-Helper2 vector generates only condition-
ally infectious particles [ 24 ]. In this case, the following additional
activation steps are required: add

3.7 Activation of
Recombinant SFV
Particles

-chymotrypsin to the virus
stocks (20 mg/mL) at a fi nal concentration of 500

α

g/mL and
incubate at room temperature for 20 min ( see Note 6 ). Stop the
reaction with Aprotinin (trypsin inhibitor, 10 mg/mL) at a fi nal
concentration of 250

μ

μ g/mL. The activated SFV particles are now

ready for infections.

3.8 Verifi cation
of Virus Titers

Due to the replication-defi cient nature of the generated virus
particles no plaques are produced, which prevents any direct titer
measurement. Instead, application of reporter genes, such as GFP
or

-galactosidase, allows titer estimation by determination of the
number of infected cells, i.e., cells expressing the respective reporter
gene. Immunofl uorescence methods can also be applied for indi-
rect titer determinations. Cell morphology characterization by
light microscopy can also give an indication of titers as SFV-infected
cells show a dramatic decrease in growth and can be easily distin-
guished from noninfected control cells through their changed
morphology (round up).

β

3.9 Visualization
of Reporter Gene
Expression

Culture BHK-21 (or other) cells to a defi ned concentration on
6- or 12-well plates or on coverslips. Infect cells with serial dilutions
(e.g., 5-fold dilutions in the range expected to give 20-50 positive
cells per microscope fi eld) of virus stocks expressing GFP or
β -galactosidase and incubate over night at 37 °C. Estimate the

titers no later than 48 h postinfection. At earlier time points the
signal, especially for some mutant vectors may be suboptimal and
at later time points the cytopathic effects result in increasing num-
bers of detached cells.

Count the number of GFP positive cells applying fl uorescence
microscopy. Approximate titers can be determined as: infectious
particles/mL based on the number of GFP positive cells per well
and taken into account the virus dilution.

3.9.1 GFP Detection