Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 223

4. To determine the number of transgene copies per cell ( a ) from
the percentage of GFP-expressing cells, the Poisson formula is
applied:

a =− ln( 1 / (( p / 100 ) − 1 ))

p = percentage of cells positive for the fl uorescent marker.

Apply the following formula to calculate the amount of
transducing units per ml ( y ):

y = 1000 * N * a / V

N = number of cells at the time of harvesting.
V = volume of vector suspension added to the well [ μ l].

The present protocol, adapted from [ 49 ], determines the infectiv-
ity of a vector suspension by comparison with a reference vector
(Fig. 1c ). The infectivity of the reference vector, which typically
encodes a fl uorescent marker, can be determined by fl ow cytome-
try according to protocol 3.2.2 .

3.2.3 Relative Measure
of Vector Infectivity by
qPCR

1. Cell infection . Plate 3 E 5 HEK 293 T cells per well in a 6-well
plate. At day 1, reduce the volume of the culture medium to
1 ml. Add 4 serial dilutions of each vector suspension, includ-
ing the reference virus (1 μ l, 1/3 μ l, 1/9 μ l, and 1/27 μ l). At

day 1, add 1 ml of fresh culture medium.
2. Isolation of genomic DNA . 48 h postinfection, harvest the cells
and proceed with isolation of cellular genomic DNA using
specifi c columns (NucleoSpin ® Tissue). Resuspend the gDNA
in H 2 O (concentration l).

3. Elimination of single-stranded DNA . Next step aims at elimi-
nating single-stranded DNA from AAV genomes that failed to
be properly processed and therefore do not contribute to
transgene expression. The total DNA extracted from infected
cells is digested for 30 min at 37 °C with Nuclease S1 at a
concentration of 54U/ml in the corresponding buffer (50 mM
NaAcetate pH 4.5, 280 mM NaCl, 4.5 mM ZnSO 4 ) ( see Note
15 ). The enzyme is inactivated for 15 min at 97 °C. Before
determination of the number of copies by qPCR, DNA is
purifi ed on NucleoSpin ® Tissue columns to eliminate the S1
digestion buffer and resuspended at ca. 5-20 ng/μl.

≥ 20 ng/ μ

l.
4. Quantitative PCR . For each sample, use two TaqMan primer
sets, one specifi c for the pAAV vector genome and one spe-
cifi c for the human albumin gene. The latter set of primers is
used to determine the amount of cellular genomes in the
qPCR reaction. Using known quantities of plasmid and/or
human genomic DNA, establish standards to determine the
number of copies for each reaction. Determine the number of

μ