Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 229

4 Notes

1. In the shuttle plasmid, the size of the whole single-stranded
packaged vector genome in between the two AAV ITRs should
be comprised between 2.5 and 5 kb for the packaging of one
complete genome per particle.
2. The pH of the HBS solution has to be precisely adjusted to
ensure high transfection effi ciency.
3. NucleoSpin ® Tissue columns for DNA purifi cation effi ciently
bind both double-stranded cell genomic DNA and viral single-
and double-stranded DNA.
4. It is usually preferred to avoid amplifi cation of a sequence
specifi c for vector genome close to the ITRs in order to avoid
interference of the amplifi cation process with the secondary
structure formed by these repeats. However, a recent report
indicates a qPCR primer set directed against the AAV2 ITR
sequence which is common to most vectors [ 50 ].
5. This protocol for anesthesia of mouse neonates is recom-
mended by the Swiss Veterinary Offi ce and can be adapted
according to local regulations for animal experimentation.
6. The Episerf medium is used to minimize contamination of the
vector preparation with serum proteins.
7. The present protocol is optimized to recover vector particles
retained within the cell cytosol or nucleus. It should be noted
that the presence of vector particles has been reported in the
cell supernatant, depending upon the AAV serotype produced
[ 51 ]. If needed, specifi c protocols can therefore be used to
maximize the yield of vector production.
8. Note that only certain AAV serotypes, such as AAV2 and
AAV6, can be purifi ed on heparin-binding columns. For most
serotypes, anion exchange chromatography is the preferred
method.
9. To avoid contaminations between viral productions, it is rec-
ommended not to regenerate the column unless the same vec-
tor has to be isolated. Use a new column for each individual
vector.
10. The vector can be stored at −80 °C in D-PBS containing Ca ++
and Mg ++ for at least 2 years. It is recommended to avoid
freeze/thaw cycles, to manipulate the vector with fi lter tips,
and to use exclusively low retention tubes.
11. In contrast to wild-type AAV, the ratio of total particles to
effective particles in recombinant vector suspensions is com-
monly found to be 100:1 [ 52 ].