FELIX Virus Production: FuGENE 6 Transfection
NB: FuGENE 6 can be obtained from Boehringer-Mannheim and has proven to be the best lipid reagent for transfection of 293T cells in our hands.

1) Split 293T cells out the day before transfection.  2-2.5 X 106 cells/6cm plate generally works well.  At the time of transfection cells should be ~50% confluent in 3mL of DMEM w/10%FCS.

2) In a sterile 1.5mL microfuge tube add 266ul of serum-free DMEM.  Add
  27ul FuGENE 6 directly to media without allowing it to touch the sides of
  the tube.

3) Incubate the FuGENE/media mixture for 5 minutes at room temperature.

4) To a second sterile 1.5mL tube add 5ug structural vector (e.g. CPR/\Env),
  3ug transfer vector (e.g. pFLX-5CL), and 2ug envelope vector (e.g.
  pCI-VSVG).

5) Dropwise add diluted FuGENE from step 3 to the tube containing the
  DNA from step 4.

6) Tap the tube to mix the contents.

7) Incubate the FuGENE/DNA mix for 15minutes at room temperature.

8) Add the mixture dropwise to the plated cells. Swirl to distribute.

9) Incubate cells at 37 degrees for 16-30hours.

10) OPTIONAL: Change media to fresh DMEM w/10% FCS.

11) Change media at 30hours post-transfection.

12) Collect supernatant at 48hrs post-transfection. (NB: supernatants can be
  collected as early as 35 hours post-transfection and as late as 72 hours and
  will still contain good titre - so you can get 2 or 3 virus collections per
  transfection, peak is somewhere between 48 and 60 hours post. Also,
  moving plates to 32degrees ~8hrs prior to collection can increase yield.)

13) Clear the supernatant by spinning in a 15mL tube at 1500RPM for 5
  minutes and/or by filtering through a .45um filter. (if you are filtering you
  will lose some titre, this loss can be minimized by pre-wetting the filter
  with serum or serum-containing media and by using low-protein binding
  filters).

14) OPTIONAL: Virus can be concentrated by centrifugation at >40000XG
  for 1.5-2hrs.  I use 50mL tubes in a fixed angle Beckman JA.20 or
  JA25.50 at 20000 RPM.  Conventionally you concentrate in an
  ultracentifuge (SW41 or SW28 rotor) at 50000XG for 1.5hrs. The pellet
  should be resuspened in TNE (Tris-NaCl-EDTA).

15) Target cells are infected by adding virus-containing supernatant or
  purified, concentrated virus diluted in media in the presence of polybrene
  (1000X stock is 5mg/mL) or protamine sulfate (1000X = 8mg/mL).  For
  best results cells should be infected in multi-well plates and then spun at
  2500RPM for 90 minutes at 30degress immediately following addition of
  virus.  Spin infection significantly enhances infectivity.

16) Place cells containing virus at 32 degrees for 6-36hrs - usual overnight
  works well.

17) Change infected cells to fresh media.

18) Analyze protein expression >48hr post-infection.

NB: This protocol can be scaled up for larger plates by multiplying ALL reagent and dilution and DNA volumes by the difference in surface area between the desired plate/flask and a 6cM plate.
 

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