generation of stable PG13 retro-producer cell lines

Protocol for the production of stable virus producing cells (PG13)

 

 day 0:

 - seed 293T cells in 60 mm plates at 1.5x10⁶ per plate in 5 ml DMEM+10%FCS

 

 day 1:

  - 2-6 hours prior to transfection: replace medium with 3 ml DMEM+10%FCS

- transfection:

- fill sterile eppendorf tube with 200 µl DMEM

- add 12 µl Fugene directly to medium

- add 2 µg expression vector DNA

- add 2 µg pCLeco DNA

- mix by gently tapping

- incubate for 15-30 minutes at room temperature

- add mixture dropwise to the cells

 

day 2:

  - Replace (already virus containing) medium of transfected cells with 3 ml DMEM+10%FCS. Desinfect used pipets in bleach, inside the flow cabinet before discarding them in the blue waste container.

- Seed PG13 cells in 12-wells plate at 1000 cells per well in 2 ml DMEM+10%FCS. For each transduction (= each construct), 3 wells of PG13 are needed.

 

 day 3:

  - remove medium from 3 wells of PG13 cells

- Add 2 ml of DMEM + 10% FCS to the second and third well of PG13

- Remove cover of one filter-package.

- collect virus containing supernatant from one 60 mm plate by suction with a 5 ml syringe (syringe without a “luer-lock”)

- Put syringe on filter by firmly pressing the syringe onto the filter, while the filter is still in its package.

- Put virus containing medium onto the first (“dry”) well of PG13 cells by carefully pressing the syringe (be careful not to put too much pressure on the filter, to prevent the filter from p(l)opping off the syringe)

- Discard syringe and filter into bottle with a little bleach, also used for pipet tips.

- Transfer 1 ml of medium from the first well of PG13 to the second well, mix carefully by pipetting up and down slowly.

- Transfer 1 ml of medium from the second well to the third well of PG13, again mix carefully.

- Transfer 1 ml of medium from the third well into a tube, add some bleach, close the tube tightly and discard into blue waste container.

- Add polybrene to the wells at 8 µg/ml. Discard pipet tips in bottle.

 

day 4:

  - Refresh medium on PG13 cells with 2 ml DMEM+10%FCS. Desinfect used pipets in bleach inside flow cabinet before discarding them in the blue waste container.

 

 day 7-8:

  - Check wells for growth and GFP-positivity

- Chose the well which is evenly transduced (GFP fluorescence on microscope or FACS, NGFR positivity on the FACS) at a high level, and which grows well. (This is not neccesserily the first well). When performing FACS analysis on PG13 producer cells, be sure to fix the cells in 1% paraformaldehyde for 30 minutes before taking them out of the room for analysis.

- Split cells when 80% confluent into T25 flasks

- (Perform transductions to check viral titer to select the best well)

- Freeze cells for later use at an early passage.

 

 

Bart-Jan Wierenga, 22-01-04

 

Revised: 25-05-2005