Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 196

Seeding of 293T cells

Day 1

Transient transfection
by calcium phosphate

Day 2

Ultracentrifuge

CaPO 2 precipitate

Harvest and concentration of lentiviral
vectors by ultracentrifugation

Day 4

Titration

Day 5

p24 titration

Titration by qPCR

Fig. 6 Schematic diagram of the various steps in lentivirus production

293T cells (ATTC-CRL-11268) are used for LV production
because of their high transfection effi ciency [ 106 , 107 ]. These
human embryonic kidney cells are immortalized with adenoviral
E1A/E1B proteins and express the SV40 large T antigen, facilitat-
ing replication of the episomal plasmid. They grow in DMEM
medium supplemented with 10 % FBS ( see Note 8 ) and 1 % strep-
tavidin/streptomycin. They are maintained in a humidifi ed incuba-
tor at 37 °C, under an atmosphere containing 5 % CO 2 . These cells
are subcultured every 3-4 days, at a dilution of 1/5 ( see Note 8).
For regular production, 5 × 10 6 cells should be plated in a 10 cm
Petri dish, whereas for large-scale production, 5.8 × 10 7 cells should
be plated in a stack, as described below (Table 1 ; see Note 9 ).

3.6.1 Day 1: Seeding
of 293T Cells

The day after plating, the cells should have reached 50-70 % con-
fl uence: smaller numbers of cells result in lower production yields.
The calcium-phosphate transfection method is used (transfection
effi ciency of up to 95 % in 293T cells). The CaPO 2 precipitate is
prepared as shown below (Table 2 ) and all the reagents are

3.6.2 Day 2: Transient
Transfection in the
Presence of Calcium
Phosphate