Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 220

and PBS. Load the vector suspension on the HPLC system
(maximal volume per loading: 9 ml). Wash with PBS until
detection is back to baseline. Elute the vector with PBS + 0.4 M
NaCl. Elution peak is observed after ca. 140 s. Collect the
eluted vector suspension as soon as UV absorbance signifi -
cantly increases and until absorbance returns to baseline.
Regenerate the column by washing it with PBS + 2 M NaCl for
10 min. The heparin column is fi nally washed with bidistilled
H 2 O and maintained in H 2 O with 1 % methanol.
7. Vector concentration and storage . The collected virus suspen-
sion is centrifuged on an Amicon Ultra-15 centrifugal fi lter
unit (cut-off 100 kDa) at 3,000 × g . Replace the buffer 3-4
times with 15 ml PBS pH 7.4. Finally, the vector suspension is
narrowed down to a volume of 250-500 l in D-PBS. If highly

concentrated vector is needed, the suspension can be further
concentrated to a fi nal volume of 50-100

μ

l using an Amicon
Ultra-4 centrifugal fi lter unit (cut-off 100 kDa). Ultimately,
the virus is resuspended in D-PBS, distributed in 10

μ

l aliquots
in low retention tubes, and stored at −80 °C ( see Note 10 ).

μ

Various methods are available to titer AAV vector suspensions (see
Fig. 1 ). The most commonly used method is to determine the
concentration of particles containing a viral genome (Fig. 1a ).
Vector concentration can easily be determined by quantitative PCR
(qPCR) and is applicable to any vector. However, this approach
does not provide any measurement of vector infectivity and there-
fore does not accurately predict the level of transgene expression
( see Note 11 ).
The infectivity of viral particles can be determined by incubating
the vector with cells permissive to viral infection. For instance, we
have determined that HEK 293 T cells can be effectively infected
by AAV6 particles ( see Note 12 ). Importantly, the vector genome
mostly remains nonintegrated in the host cellular genome and is
therefore not replicated during cell division. Therefore, the number
of transgene copies per cell decreases over time in mitotic cell lines
( see Note 13 ).
Flow cytometry provides an accurate estimate of transgene
expression for vectors encoding fl uorescent marker proteins. In
particular, the percentage of cells with detectable expression of the
fl uorescent protein is in direct proportion to the number of trans-
ducing units (TUs) (Fig. 1b ). Alternatively, qPCR can measure the
number of transgene copies delivered by viral particles, with respect
to the number of cells, as determined by the amount of cell
genomes present in the reaction (Fig. 1c ). However, in most cases,
these methods reliably provide only relative measures of infectivity
between vector preparations. Therefore, it is useful to establish
standard vector preparations within labs, or available through com-
mercial sources, that can be used for comparison of infectivity

3.2 AAV6 Vector
Titration and Quality
Control