Biology Reference
In-Depth Information
5.3 Generation
of Lentiviral Particles
This protocol is designed for virus production in 10 cm culture
plates or T-75 cm 2 fl asks.
1. (Day 1) Trypsinize HEK 293T (packaging cells), count these
cells and add 3×10 6 HEK 293T cells to a 10 cm culture plate
in 10 mL of pre-warmed medium ( see Note 16 ).
2. Cell density should be approx. 50 % confl uent for transfection
( see Note 17 ).
3. (Day 2) Remove 2× HBS buffer and vectors from −20 °C
freezer and pre-warm at room temperature.
4. Prepare 1 mL of calcium phosphate-DNA suspension for each
10 cm plate of cells as follows: Mix 12
g of the transfer vector
pLKO1.puro or pLJM1 containing the transgene ( see Note
18 ), 12
μ
g of pCMV-VSV-G or
pHit/G and fi ll it up with water to a total volume of 438
μ
g of pCMV
R8.2 and 6
μ
μ
L.
Then add 62
μ
L of 2 M CaCl2 and mix by pipetting. Next add
L of 2× HBS dropwise while holding the solution
motionless, then incubate the mixture at room temperature for
10 min.
5. Mix 12
500
μ
g of the transfer vector pLKO1.puro or pLJM1 con-
taining the transgene ( see Note 18 ), 12
μ
μ
g of pCMV
R8.2
and 6
g of pCMV-VSV-G or pHit/G and fi ll it up with water
to a total volume of 438
μ
μ
L. Then add 62
μ
L of 2 M CaCl 2 and
L of 2× HBS dropwise while hold-
ing the solution motionless and incubate the mixture at room
temperature for 10 min.
6. Add the 1 mL of calcium phosphate-DNA suspension dropwise
into the medium of the HEK 293T cells ( see Notes 19 and 20 ).
7. Incubate the cells at 37 °C, 10 % CO 2 .
8. (Day 3) After 14-16 h, discard the culture medium from the
HEK 293T cells and replace with 11 mL pre-warmed fresh
medium to produce virions overnight. The transfection effi -
ciency can be assessed by fl uorescence microscopy of packag-
ing cells transfected with the GFP-expressing control vector.
9. Plate an appropriate number of target cells, so that confl uence
reaches 50-60 % the next day. For 10 cm plates the cell num-
ber should be about 1×10 6 , for 6 wells 1×10 5 cells are
suffi cient.
10. (Day 4) Remove the 11 mL virus-containing medium from the
HEK 293T cells by a 20 mL syringe and fi lter into a 50 mL
tube through a sterile fi lter (pore size 0.45
mix by pipetting. Add 500
μ
m) to remove cell
debris. To enhance infection, add polybrene in a fi nal concen-
tration of 4
μ
g/mL ( see Note 21 ).
11. Dispose the packaging cells.
μ
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