Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 58

Remember always to check pH, adjust accordingly, and fi lter
sterilize if opened outside a sterile tissue culture hood.
5. Small loss of producer cells upon medium replacement will fur-
ther result in decrease in titer in time. Be careful when replac-
ing medium in order to avoid detaching cells. If too many cells
detach during the induction step, collect cells from each plate
in a separate Falcon tube, centrifuge to pellet the cells, resus-
pend in fresh medium and return them to the dish where they
originally come from. This will minimize the loss of your pro-
ducer cells and can partially save some virus produced.
6. It is important to perform this fi ltration step, as viral capsid
debris can be toxic when virus applied to target cells. You can
also use a 0.22 m fi lter to obtain purer sample.

7. Titers can drop as much as 2-4-fold with each freeze-thaw
cycle [ 128 , 129 ].
8. Add the remaining viral supernatant slowly, by releasing it to
the wall of the tube to avoid disrupting the sucrose cushion.
9. It is recommended to keep the tubes still while setting the
cushion.
10. The percentage of gated cells in negative control should be
less than 0.1 %.
11. Viral titers will vary depending on the cell line used for titra-
tion. When using HEK 293T cells, titers for concentrated
preps are expected to be within the range of 1 × 10 9 -10 10 TU/
ml. In our lab, with the production method described in this
chapter, we have managed to achieve titers of 1 × 10 11 . When
using HeLa cells, titers are generally tenfold lower [ 106 ]. In
any occasion, if the titer of your concentrated stock is lower
than 1 × 10 8 TU/ml, we strongly recommend producing a
new lentiviral stock.
12. p24 ELISA kits such as the one from Zeptometrix contain
inactivated wild-type HIV-1, which poses a potential safety
risk. For this reason it is recommended to perform under
Biosafety Level 2 (BL2) condition.
13. If you obtain lower than expected functional vector titers upon
titration using p24 ELISA, it is possible that the p24 is not
associated with functional vector particles.
14. For RNA work, be extremely clean and careful. Use a dedi-
cated area and a dedicated set of pipettes for RNA isolation.
Treat surface and pipettes with RNase Away sprays and keep
away any instrumentation used for DNA work. Remember to
spray your hands with RNase Away. Ideally RNA isolation and
qPCR setup should be performed in a dedicated hood.
15. When setting up qPCR reactions keep your plate on ice, per-
form each reaction at least in duplicate.

ʼ