generation of lentiviral particles

Lentiviralproduction protocol:

 

Day 1

- Coat dishes for two hours with 0.1% gelatin (fridge)

- Plate 5 ( 1,5 tot 5) million293T cells in 10mL DMEM + 10% FCS into a gelatin coated 10 cm dish per group, incubate ON 37ºC, 5% CO2

 

Day 2

- transfect with Fugene:

Tube 1:

- DMEM (–FCS)                                                100 µl

- packaging construct (pCMV Δ8.91)                 3µg

- glycoprotein envelop plasmid (VSV-G)            0.7 µg

- vector construct (pTRIP)                                 3 µg

 

Tube 2:

- DMEM (–FCS)                                                400 µl

- Fugene                                                          21µl

(in medium, not against the edges of the tube)

 

- Add tube 1 to tube 2, flick gentle and allow complex formation for 20 minutes at RT

- Add mixture dropwise to 293T cells, swirl real gentle, incubate ON 37ºC, 5% CO2

 

Day 3

- Change medium of 293T cells to 5 mL HPGM and incubate ON 37ºC, 5% CO2

 

Day 4

- Remove 5 mL medium from 293T cells (very gentle, a lot of the cells float, since 293T cells don’t like HPGM) into a 12 ml tube.

- Pass over two filters (low protein binding filters) to remove residual 293T cells (first time, the filter might clog and snap, since a lot of 293T cells are also in this medium, hence the second filter step with new filter) (This is less pronounced when using gelatin coated dishes)

- freeze virus containing supernatant in aliquots of 500 µl in cryotubes in -80ºC and use when necessary.