Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 101

Cells are washed with PBS and fi xed in cold methanol at −20 °C
for 5 min. Wash cells 3 times with PBS and stain for at least 2 h in
staining solution at 37 °C or room temperature. Count the X-gal
(blue) positive cells and estimate the titers as for GFP detection.

3.9.2 X-gal Staining

Cells on coverslips are rinsed twice with PBS and fi xed for 6 min at
−20 °C in methanol. Wash cells 3 times in PBS. Incubate for
30 min at room temperature in PBS containing 0.5 % gelatin and
0.25 % BSA to prevent unspecifi c binding. Replace the blocking
buffer with primary antibody in the same buffer for 30 min at
room temperature. Wash 3 times with PBS and incubate with sec-
ondary antibody for 30 min at room temperature. Wash 3 times
with PBS and once with H 2 O, and air dry coverslips. Mount on
glass slides using 10

3.9.3 Immuno-
fl uorescence

L Moviol 4-88 containing 2.5 % DABCO
(1,4-diazobicyclo-[2.2.2]-octane). Count the positive cells and
estimate the titers as for GFP detection.

μ

It is advisable to rapidly evaluate the transgene expression from
generated virus stocks before any more detailed and large-scale
expression is preformed. It also allows to determining the best
expression conditions. Factors of importance for expression opti-
mization are virus concentration, host cell line, and time of cell
harvest. The size of the gene product and expression levels can be
obtained by Western blotting. It requires available antibodies
against the target protein or introduction of tags in the vector con-
structs. Alternatively, cells can be metabolically labeled with
35 S-methionine as a means to verify transgene expression (Fig. 3 ).

3.10 Evaluation
of Gene Expression

Infect appropriate cells (BHK-21, CHO-K1, and HEK293) cul-
tured on 6-, 12-, or 24-well plates with serial dilutions of virus
stocks. Add 250, 125, and 62.5

3.10.1 Western Blotting

L lysis buffer per 6-, 12-, and
24-well plate, respectively, at various times postinfection and incu-
bate 10 min on ice. Resuspend the cells thoroughly and load
samples on 10-12 % SDS-PAGE. Transfer protein material for 30
min to a Hybond ECL nitrocellulose fi lters. Treat the fi lters with
5 % milk in TBST at +4 °C for 30 min followed by primary anti-
body at room temperature for 30 min. After treatment with the
secondary antibody at room temperature for 30 min, visualize
specifi c bands with the ECL Chemiluminescence kit.

μ

Infect alternative cells (BHK-21, CHO-K1, and HEK293) cultured
on 6-, 12-, or 24-well plates with serial dilutions of virus stocks.
Remove medium, wash cells once with PBS and add Starvation
medium and incubate at 37 °C for 30 min. Replace medium with
50-100

3.10.2 Metabolic
Labeling

Ci/mL of 35 S-methionine (in Starvation medium) and
incubate 20 min at 37 °C. Remove medium, wash cells twice with
PBS and add Chase medium for appropriate time (15 min to 3 h).
Remove Chase medium and wash once with PBS. Add 250

μ

μ L lysis