Lentiviral Vectors as Research Tools in Neurobiology: Design and Production - Viral Vector Approaches in Neurobiology and Brain Diseases - page 7

3. After 5 days, collect the cells, wash in PBS with 1 % FBS, and
resuspend in 2 % paraformaldehyde in PBS 1×.
4. The titer (transducing units, T.U.) is determined by using
Accuri C6 Flow Cytometer (BD Bioscience) and counting the
percentage of GFP-positive cells in each dilution. When the
percentage is between 2.5 % and 25 %, the titer can be deter-
mined using the following formula:

510 4 percentage of GFPpositivecells × cells plated the first day ( × )

TiterTUml (../ ) =

dilution

LVs are stable at −80 °C for at least 6 months. Thaw aliquots on wet
ice. For reliable results is not recommended to refreeze aliquots
after thawing: in our hand we notice a decrease of the titer of at least
one order of magnitude after a freeze-thaw cycle. The transduction
of cell in cultures is very simple: just add the LV suspension to the
desired medium. To choose the correct amount of LV for each
experiment is important to value the desired multiplicity of infec-
tion (MOI). MOI is defined as the ratio between the number of
TU and the number of cell to be transduced. The right MOI
depends very much on the application. As a general guideline,
low MOI (around 0.25) guarantees that virtually every cell receive
only one copy of the transgene, whereas high MOI (from 3 to 5)
guarantees that virtually 100 % of the cells will express in one or
more copy the transgene. For in vivo use (e.g., stereotaxic injection
in brain), the use of LVs under the titer of 10 9 is not recommend.

4.3 Use of LVs

5 Notes

1. The production and the use of LVs are biohazardous.
According to the law, all the procedures must be performed on
facilities at biosafety level 2 or more.
2. The protocol provided refers to self-inactivating, third-generation
vectors [ 11 ]. The plasmids used for packaging, kindly provided
by Luigi Naldini, were pREV (expressing REV protein), pVSVG
(expressing the envelope: VSV-G), and pRRE (expressing capsid,
polymerase, protease, and integrase proteins).

References

1. Knipe D, Howley P (eds) (2006) Fields virol-
ogy, 5th edn. Wolters Kluwer, Philadelphia PA
2. Buchschacher GL Jr, Panganiban AT (1992)
Human immunodeficiency virus vectors for
inducible expression of foreign genes. J Virol
66(5):2731-2739
3. Parolin C, Dorfman T, Palu G, Gottlinger H,
Sodroski J (1994) Analysis in human immuno-

deficiency virus type 1 vectors of cis-acting
sequences that affect gene transfer into human
lymphocytes. J Virol 68(6):3888-3895
4. Poznansky M, Lever A, Bergeron L, Haseltine
W, Sodroski J (1991) Gene transfer into
human lymphocytes by a defective human
immunodeficiency virus type 1 vector. J Virol
65(1):532-536