Biomedical Engineering Reference
In-Depth Information
3
Methods
3.1 Lentiviral Vector
Production
1. Subclone the cDNAs for known or candidate oncogenes into
pLVX-Puro-CMV (Clontech) (
see
Note 1
).
2. Prepare transfection materials. Approximately 24 h before
transfection, seed 4-5 × 10
6
Lenti-X 293T cells/100 mm plate,
in 10 ml of growth medium. Make sure that the cells are plated
evenly. Incubate at 37 °C, 5 % CO
2
overnight. Continue to
incubate the cells until you are ready to add the transfection
mixture in step 8. The cells should be 80-90 % confl uent at the
time of transfection.
3. Thoroughly vortex Xfect polymer (Lenti-X Packaging System
from Clontech, cat# 631247).
4. For each transfection sample, prepare two microcentrifuge
tubes by adding reagents in the following order:
(a) Tube 1 (plasmid DNA)
557
ʼ
l Xfect reaction buffer
●
36
ʼ
l Lenti-X HTX packaging mix
●
7
ʼ
l Lenti-X vector DNA (1
ʼ
g/
ʼ
l)
●
l total volume
(b) Tube 2 (polymer)
600
ʼ
●
592.5
ʼ
l Xfect reaction buffer
●
7.5
ʼ
l Xfect polymer
●
l total volume
5. Vortex each tube well to mix.
6. Add the polymer solution (tube 2) to the plasmid DNA solution
(tube 1), and vortex well at a medium speed for 10 s.
600
ʼ
●
7. Incubate each DNA-Xfect mixture for 10 min at room tem-
perature to allow nanoparticle complexes to form.
8. Add the entire 1,200
l of DNA-Xfect solution dropwise to
the cell culture medium from step 2. Rock the plate gently
back and forth to mix.
9. Incubate the plate at 37 °C.
10. After 4 h to overnight, replace the transfection medium with
10-ml fresh complete growth medium (containing Tet System
Approved FBS), and incubate at 37 °C for an additional
24-48 h (
see
Note 2
). Harvest the lentiviral supernatants and
pool similar stocks, if desired. Centrifuge briefl y (500 ×
g
for
10 min) or fi lter through a 0.45
ʼ
ʼ
m fi lter to remove cellular
debris (
see
Note 3
).
Search WWH ::
Custom Search