Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 146

strip of paper on the floor of the runway and allowing the rat to
walk into the escape box. The footprint patterns are assessed quan-
titatively by measurements of forepaw and hindpaw stride length,
base width, and forepaw/hindpaw footprint overlap. The mean
values from each set of six steps are calculated.

Rats' locomotor coordination is assessed on an accelerating
rotarod. Rats are placed on the rotarod which accelerates from 4 to
40 rpm over 5 min. Rats are given two training sessions and one
trial per day over 3 days when the latency to fall is recorded.

3.5.3 Rotarod
Performance

4 Notes

1. The individual chemicals used to prepare the 2× HeBs buffer
should be accurately weighed as slight variations between
batches can affect transfection.
2. We maintain a twice-weekly splitting and cell plating routine
on a Monday and Thursday cycle, with cells split 1:5 on
Monday and 1:6 on Thursday. Cell density at the time of trans-
fection and transfection efficiency can have a major influence
on the resulting vector titers. We typically aim for ~70 % con-
fluency at the time of transfection to ensure there will be suf-
ficient numbers of transfected cells that will package vector,
taking into account that the cell growth is maintained for 3
days following transfection.
3. We have also packaged vectors by plating the cells directly into
IMDM at a ratio of one fully confluent T175 flask to 3-3.5
15 cm dishes. This alleviates the need for step 2 in Sect. 3.1.2 .
4. The AAV cis plasmid consists of transgene expression cassette
flanked by AAV2 inverted terminal repeats. AAV serotype, for
example, AAV1, 4, 5, 8, and 9, rh 10, is determined by the
inclusion of a plasmid expressing serotype-specific capsid pro-
teins. As AAV2 inverted terminal repeats are utilized, these
vectors are often referred to as pseudotyped AAV vectors in the
literature. High-quality transfection-grade plasmid is required
for AAV packaging. We typically use plasmid purification kits
sourced from Qiagen or Invitrogen.
5. While the method we have provided specifies adding an equal
volume of 2× HeBs buffer to the DNA transfection mix, we
advise testing each batch of 2× HeBs buffer to determine the
volume required for optimal transfection and rAAV packaging.
This can be achieved by transfecting 15 cm dishes of cells with
the equivalent of 6-13 mL 2× HeBs/5 plate transfection mix
using a fluorescent reporter gene-containing construct. Follow
Sect. 3.1.2 , steps 3-5. Sixteen hours after transfection, note
the size of the calcium phosphate/DNA precipitate. The pre-
cipitate should be very fine and just visible to the naked eye.